Methods for increasing hydrolysis of cellulosic material

ABSTRACT

The present invention relates to methods for pretreating a cellulosic material, methods for degrading or converting a cellulosic material, methods for producing a fermentation product, and methods of fermenting a cellulosic material under anaerobic conditions.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/139,412, filed Dec. 19, 2008, which application is incorporatedherein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing filed electronically byEFS, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods for increasing hydrolysis ofcellulosic material with an enzyme composition.

2. Description of the Related Art

Cellulose is a polymer of the simple sugar glucose linked bybeta-1,4-bonds. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiose is awater-soluble beta-1,4-linked dimer of glucose. Beta-glucosidaseshydrolyze cellobiose to glucose.

It is well known in the art that oxidation of biomolecules such as DNA,lipids, or protein is a significant issue in biological systems.Consequently, removal or exclusion of reactive oxygen species mayimprove the performance of cellulose-hydrolyzing enzyme systems.

Hydrolysis of cellulosic material, e.g., lignocellulose, by an enzymecomposition can be reduced by oxidative damage to components of theenzyme composition and/or oxidation of the cellulosic material by, forexample, molecular oxygen.

The present invention provides methods for increasing hydrolysis ofcellulosic materials with an enzyme composition.

SUMMARY OF THE INVENTION

The present invention relates to methods for pretreating a cellulosicmaterial, comprising: pretreating the cellulosic material underanaerobic conditions.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition under anaerobic conditions.

The present invention also relates to methods for producing afermentation product, comprising:

(a) saccharifying a cellulosic material with an enzyme composition underanaerobic conditions;

(b) fermenting the saccharified cellulosic material with one or morefermenting microorganisms to produce the fermentation product; and

(c) recovering the fermentation product from the fermentation.

The present invention further relates to methods of fermenting acellulosic material, comprising: fermenting the cellulosic material withone or more fermenting microorganisms, wherein the cellulosic materialis hydrolyzed with an enzyme composition under anaerobic conditions.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show reduced cellobiose dehydrogenase (CBDH) inhibitionof pretreated corn stover (PCS) hydrolysis by a Trichoderma reeseicellulase composition under anaerobic conditions. Panel A: fractionalcellulose conversion is plotted for various hydrolysis conditions. Solidbars: anaerobic hydrolysis; hatched bars: aerobic hydrolysis. Panel B:fractional cellulose conversion is plotted for cellobiose dehydrogenaseinhibition of the Trichoderma reesei cellulase composition underanaerobic (open circles) and aerobic (closed circles) conditions.

FIG. 2 shows a comparison of aerobic and anaerobic hydrolysis of PCS atvarious concentrations of a Trichoderma reesei cellulase composition.Circles: 1 day hydrolysis; squares: 4 day hydrolysis; diamonds: 7 dayhydrolysis; open symbols: anaerobic hydrolysis; closed symbols: aerobichydrolysis.

FIG. 3 shows a comparison of aerobic and anaerobic hydrolysis of AVICEL®by a Trichoderma reesei cellulase composition, as well as ferrous ironinhibition of the Trichoderma reesei cellulase composition under aerobicand anaerobic hydrolysis conditions.

DEFINITIONS

Anaerobic conditions: The term “anaerobic conditions” is defined hereinas processing conditions in the absence of molecular oxygen, or at anoxygen level significantly lower than those from ambient, atmosphericconditions (approximately 0.2 atmosphere pressure of oxygen gas orapproximately 0.3 mM water-dissolved oxygen).

In one aspect, the oxygen level is preferably no greater than 0.15, morepreferably no greater than 0.1, more preferably no greater than 0.05,more preferably no greater than 0.02, even more preferably no greaterthan 0.01, and most preferably no greater than 0.002 atmosphere pressureof oxygen gas. In another aspect, the oxygen level is preferably nogreater than 0.15 atmosphere pressure of oxygen gas. In another aspect,the oxygen level is preferably no greater than 0.1 atmosphere pressureof oxygen gas. In another aspect, the oxygen level is preferably nogreater than 0.05 atmosphere pressure of oxygen gas. In another aspect,the oxygen level is no greater than 0.02 atmosphere pressure of oxygengas. In another aspect, the oxygen level is 0.01 atmosphere pressure ofoxygen gas. In another aspect, the oxygen level is no greater than 0.002atmosphere pressure of oxygen gas.

In another aspect, the oxygen level is preferably no greater than 0.25mM, more preferably no greater than 0.2 mM, more preferably no greaterthan 0.15 mM, more preferably no greater than 0.1 mM, more preferably nogreater than 0.05 mM, more preferably no greater than 0.025 mM, morepreferably no greater than 0.01 mM, even more preferably no greater0.002 mM, and most preferably no greater than 0.001 mM water-dissolvedoxygen. In another aspect, the oxygen level is preferably no greaterthan 0.25 mM water-dissolved oxygen. In another aspect, the oxygen levelis preferably no greater than 0.2 mM water-dissolved oxygen. In anotheraspect, the oxygen level is preferably no greater than 0.15 mMwater-dissolved oxygen. In another aspect, the oxygen level ispreferably no greater than 0.1 mM water-dissolved oxygen. In anotheraspect, the oxygen level is preferably no greater than 0.05 mMwater-dissolved oxygen. In another aspect, the oxygen level ispreferably no greater than 0.025 mM water-dissolved oxygen. In anotheraspect, the oxygen level is preferably no greater than 0.01 mMwater-dissolved oxygen. In another aspect, the oxygen level ispreferably no greater than 0.002 mM water-dissolved oxygen. In anotheraspect, the oxygen level is preferably no greater than 0.001 mMwater-dissolved oxygen.

Cellobiose dehydrogenase: The term “cellobiose dehydrogenase” is definedherein as a cellobiose:acceptor 1-oxidoreductase (E.C. 1.1.99.18) thatcatalyzes the conversion of cellobiose in the presence of an acceptor tocellobiono-1,5-lactone and a reduced acceptor.2,6-Dichlorophenolindophenol can act as acceptor, as can iron,especially Fe(SCN)₃, molecular oxygen, ubiquinone, or cytochrome C, andlikely many other polyphenolics. Substrates of the enzyme arecellobiose, cello-oligosaccharides, lactose, andD-glucosyl-1,4-β-D-mannose.

Cellobiose dehydrogenases comprise two families, 1 and 2, differentiatedby the presence of a cellulose binding motif (CBM). The 3-dimensionalstructure of cellobiose dehydrogenase features two globular domains,each containing one of two cofactors: a heme or a flavin. The activesite lies at a cleft between the two domains. The catalytic cycle ofcellobiose dehydrogenase follows an ordered sequential mechanism.Oxidation of cellobiose occurs via 2-electron transfer from cellobioseto the flavin, generating cellobiono-1,5-lactone and reduced flavin. Theactive FAD is regenerated by electron transfer to the heme group,leaving a reduced heme. The native state heme is regenerated by reactionwith the oxidizing substrate at the second active site.

The oxidizing substrate is preferentially iron ferricyanide, cytochromeC, or an oxidized phenolic compound such as dichloroindophenol (DCIP), asubstrate commonly used for colorimetric assays. Metal ions and O₂ arealso substrates, but for most cellobiose dehydrogenases the reactionrate for these substrates is several orders of magnitude lower than thatobserved for iron or organic oxidants. Following cellobionolactonerelease, the product may undergo spontaneous ring-opening to generatecellobionic acid (Hallberg et al., 2003, J. Biol. Chem. 278: 7160-7166).

Cellulolytic activity: The term “cellulolytic activity” is definedherein as a biological activity that hydrolyzes a cellulosic material.The two basic approaches for measuring cellulolytic activity include:(1) measuring the total cellulolytic activity, and (2) measuring theindividual cellulolytic activities (endoglucanases, cellobiohydrolases,and beta-glucosidases) as reviewed in Zhang et al., Outlook forcellulase improvement: Screening and selection strategies, 2006,Biotechnology Advances 24: 452-481. Total cellulolytic activity isusually measured using insoluble substrates, including Whatman No 1filter paper, microcrystalline cellulose, bacterial cellulose, algalcellulose, cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman No 1filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (chose, 1987,Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic activity isdetermined by measuring the increase in hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-20mg of cellulolytic protein/g of cellulose in PCS for 3-7 days at 50-65°C. compared to a control hydrolysis without addition of cellulolyticprotein. Typical conditions are 1 ml reactions, washed or unwashed PCS,5% insoluble solids, 50 mM sodium acetate pH 5, 1 mM MnSO₄, 50-65° C.,72 hours, sugar analysis by AMINEX® HPX-87H column (Bio-RadLaboratories, Inc., Hercules, Calif., USA).

Endoglucanase: The term “endoglucanase” is defined herein as anendo-1,4-(1,3; 1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4),which catalyses endohydrolysis of 1,4-beta-D-glycosidic linkages incellulose, cellulose derivatives (such as carboxymethyl cellulose andhydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixed beta-1,3glucans such as cereal beta-D-glucans or xyloglucans, and other plantmaterial containing cellulosic components. Endoglucanase activity can bedetermined based on a reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) hydrolysis according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268.

Cellobiohydrolase: The term “cellobiohydrolase” is defined herein as a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91), which catalyzes thehydrolysis of 1,4-beta-D-glucosidic linkages in cellulose,cellooligosaccharides, or any beta-1,4-linked glucose containingpolymer, releasing cellobiose from the reducing or non-reducing ends ofthe chain (Teeri, 1997, Crystalline cellulose degradation: New insightinto the function of cellobiohydrolases, Trends in Biotechnology 15:160-167; Teeri et al., 1998, Trichoderma reesei cellobiohydrolases: whyso efficient on crystalline cellulose?, Biochem. Soc. Trans. 26:173-178). For purposes of the present invention, cellobiohydrolaseactivity is determined using a fluorescent disaccharide derivative4-methylumbelliferyl-β-D-lactoside according to the procedures describedby van Tilbeurgh et al., 1982, FEBS Letters 149: 152-156 and vanTilbeurgh and Claeyssens, 1985, FEBS Letters 187: 283-288.

Beta-glucosidase: The term “beta-glucosidase” is defined herein as abeta-D-glucoside glucohydrolase (E.C. 3.2.1.21), which catalyzes thehydrolysis of terminal non-reducing beta-D-glucose residues with therelease of beta-D-glucose. For purposes of the present invention,beta-glucosidase activity is determined according to the basic proceduredescribed by Venturi et al., 2002, Extracellular beta-D-glucosidase fromChaetomium thermophilum var. coprophilum: production, purification andsome biochemical properties, J. Basic Microbiol. 42: 55-66. One unit ofbeta-glucosidase activity is defined as 1.0 μmole of p-nitrophenolproduced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 100 mM sodiumcitrate containing 0.01% TWEEN® 20.

Cellulolytic enhancing activity: The term “cellulolytic enhancingactivity” is defined herein as a biological activity that enhances thehydrolysis of a cellulosic material by polypeptides having cellulolyticactivity. For purposes of the present invention, cellulolytic enhancingactivity is determined by measuring the increase in reducing sugars orthe increase of the total of cellobiose and glucose from the hydrolysisof a cellulosic material by cellulolytic protein under the followingconditions: 1-50 mg of total protein/g of cellulose in PCS, whereintotal protein is comprised of 50-99.5% w/w cellulolytic protein and0.5-50% w/w protein of cellulolytic enhancing activity for 1-7 day at50-65° C. compared to a control hydrolysis with equal total proteinloading without cellulolytic enhancing activity (1-50 mg of cellulolyticprotein/g of cellulose in PCS). In a preferred aspect, a mixture ofCELLUCLAST® 1.5 L (Novozymes A/S, Bagsværd, Denmark) in the presence of3% of total protein weight Aspergillus oryzae beta-glucosidase(recombinantly produced in Aspergillus oryzae according to WO 02/095014)or 3% of total protein weight Aspergillus fumigatus beta-glucosidase(recombinantly produced in Aspergillus oryzae as described in WO2002/095014) of cellulase protein loading is used as the source of thecellulolytic activity.

The polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a cellulosic material catalyzed by proteins havingcellulolytic activity by reducing the amount of cellulolytic enzymerequired to reach the same degree of hydrolysis preferably at least1.01-fold, more preferably at least 1.05-fold, more preferably at least1.10-fold, more preferably at least 1.25-fold, more preferably at least1.5-fold, more preferably at least 2-fold, more preferably at least3-fold, more preferably at least 4-fold, more preferably at least5-fold, even more preferably at least 10-fold, and most preferably atleast 20-fold.

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase”or “Family GH61” is defined herein as a polypeptide falling into theglycoside hydrolase Family 61 according to Henrissat B., 1991, Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities, Biochem. J. 280: 309-316, and Henrissat B., and BairochA., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696. Presently, Henrissat lists theGH61 Family as unclassified indicating that properties such asmechanism, catalytic nucleophile/base, and catalytic proton donors arenot known for polypeptides belonging to this family.

Xylan degrading activity: The terms “xylan degrading activity” or“xylanolytic activity” are defined herein as a biological activity thathydrolyzes xylan-containing material. The two basic approaches formeasuring xylanolytic activity include: (1) measuring the totalxylanolytic activity, and (2) measuring the individual xylanolyticactivities (endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, Recent progress in the assays of xylanolytic enzymes, 2006,Journal of the Science of Food and Agriculture 86(11): 1636-1647;Spanikova and Biely, 2006, Glucuronoyl esterase—Novel carbohydrateesterase produced by Schizophyllum commune, FEBS Letters 580(19):4597-4601; Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek,1997, The beta-D-xylosidase of Trichoderma reesei is a multifunctionalbeta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including oat spelt,beechwood, and larchwood xylans, or by photometric determination of dyedxylan fragments released from various covalently dyed xylans. The mostcommon total xylanolytic activity assay is based on production ofreducing sugars from polymeric 4-O-methyl glucuronoxylan as described inBailey, Biely, Poutanen, 1992, Interlaboratory testing of methods forassay of xylanase activity, Journal of Biotechnology 23(3): 257-270.

For purposes of the present invention, xylan degrading activity isdetermined by measuring the increase in hydrolysis of birchwood xylan(Sigma Chemical Co., Inc., St. Louis, Mo., USA) by xylan-degradingenzyme(s) under the following typical conditions: 1 ml reactions, 5mg/ml substrate (total solids), 5 mg of xylanolytic protein/g ofsubstrate, 50 mM sodium acetate pH 5, 50° C., 24 hours, sugar analysisusing p-hydroxybenzoic acid hydrazide (PHBAH) assay as described byLever, 1972, A new reaction for colorimetric determination ofcarbohydrates, Anal. Biochem 47: 273-279.

Xylanase activity: The term “xylanase activity” is defined herein as a1,4-beta-D-xylan-xylohydrolase activity (E.C. 3.2.1.8) that catalyzesthe endo-hydrolysis of 1,4-beta-D-xylosidic linkages in xylans. Forpurposes of the present invention, xylanase activity is determined usingbirchwood xylan as substrate. One unit of xylanase activity is definedas 1.0 μmole of reducing sugar (measured in glucose equivalents asdescribed by Lever, 1972, A new reaction for colorimetric determinationof carbohydrates, Anal. Biochem 47: 273-279) produced per minute duringthe initial period of hydrolysis at 50° C., pH 5 from 2 g of birchwoodxylan per liter as substrate in 50 mM sodium acetate containing 0.01%TWEEN® 20.

Beta-xylosidase activity: The term “beta-xylosidase activity” is definedherein as a beta-D-xyloside xylohydrolase (E.C. 3.2.1.37) that catalyzesthe exo-hydrolysis of short beta (1→4)-xylooligosaccharides, to removesuccessive D-xylose residues from the non-reducing termini. For purposesof the present invention, one unit of beta-xylosidase activity isdefined as 1.0 μmole of p-nitrophenol produced per minute at 40° C., pH5 from 1 mM p-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodiumcitrate containing 0.01% TWEEN® 20.

Acetylxylan esterase activity: The term “acetylxylan esterase activity”is defined herein as a carboxylesterase activity (EC 3.1.1.72) thatcatalyses the hydrolysis of acetyl groups from polymeric xylan,acetylated xylose, acetylated glucose, alpha-napthyl acetate, andp-nitrophenyl acetate. For purposes of the present invention,acetylxylan esterase activity is determined using 0.5 mMp-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0containing 0.01% TWEEN™ 20. One unit of acetylxylan esterase activity isdefined as the amount of enzyme capable of releasing 1 μmole ofp-nitrophenolate anion per minute at pH 5, 25° C.

Feruloyl esterase activity: The term “feruloyl esterase activity” isdefined herein as a 4-hydroxy-3-methoxycinnamoyl-sugar hydrolaseactivity (EC 3.1.1.73) that catalyzes the hydrolysis of the4-hydroxy-3-methoxycinnamoyl (feruloyl) group from an esterified sugar,which is usually arabinose in “natural” substrates, to produce ferulate(4-hydroxy-3-methoxycinnamate). Feruloyl esterase is also known asferulic acid esterase, hydroxycinnamoyl esterase, FAE-III, cinnamoylester hydrolase, FAEA, cinnAE, FAE-I, or FAE-II. For purposes of thepresent invention, feruloyl esterase activity is determined using 0.5 mMp-nitrophenylferulate as substrate in 50 mM sodium acetate pH 5.0. Oneunit of feruloyl esterase activity equals the amount of enzyme capableof releasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25°C.

Alpha-glucuronidase activity: The term “alpha-glucuronidase activity” isdefined herein as an alpha-D-glucosiduronate glucuronohydrolase activity(EC 3.2.1.139) that catalyzes the hydrolysis of an alpha-D-glucuronosideto D-glucuronate and an alcohol. For purposes of the present invention,alpha-glucuronidase activity is determined according to de Vries, 1998,J. Bacteriol. 180: 243-249. One unit of alpha-glucuronidase activityequals the amount of enzyme capable of releasing 1 μmole of glucuronicor 4-O-methylglucuronic acid per minute at pH 5, 40° C.

Alpha-L-arabinofuranosidase activity: The term“alpha-L-arabinofuranosidase activity” is defined herein as analpha-L-arabinofuranoside arabinofuranohydrolase activity (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeactivity acts on alpha-L-arabinofuranosides, alpha-L-arabinanscontaining (1,3)- and/or (1,5)-linkages, arabinoxylans, andarabinogalactans. Alpha-L-arabinofuranosidase is also known asarabinosidase, alpha-arabinosidase, alpha-L-arabinosidase,alpha-arabinofuranosidase, polysaccharide alpha-L-arabinofuranosidase,alpha-L-arabinofuranoside hydrolase, L-arabinosidase, oralpha-L-arabinanase. For purposes of the present invention,alpha-L-arabinofuranosidase activity is determined using 5 mg of mediumviscosity wheat arabinoxylan (Megazyme International Ireland, Ltd.,Bray, Co. Wicklow, Ireland) per ml of 100 mM sodium acetate pH 5 in atotal volume of 200 μl for 30 minutes at 40° C. followed by arabinoseanalysis by AMINEX® HPX-87H column chromatography (Bio-Rad Laboratories,Inc., Hercules, Calif., USA).

Cellulosic material: The cellulosic material can be any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, herbaceous material,agricultural residue, forestry residue, municipal solid waste, wastepaper, and pulp and paper mill residue (see, for example, Wiselogel etal., 1995, in Handbook on Bioethanol (Charles E. Wyman, editor), pp.105-118, Taylor & Francis, Washington D.C.; Wyman, 1994, BioresourceTechnology 50: 3-16; Lynd, 1990, Applied Biochemistry and Biotechnology24/25: 695-719; Mosier et al., 1999, Recent Progress in Bioconversion ofLignocellulosics, in Advances in Biochemical Engineering/Biotechnology,T. Scheper, managing editor, Volume 65, pp. 23-40, Springer-Verlag, NewYork). It is understood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In a preferred aspect, thecellulosic material is lignocellulose.

In one aspect, the cellulosic material is herbaceous material. Inanother aspect, the cellulosic material is agricultural residue. Inanother aspect, the cellulosic material is forestry residue. In anotheraspect, the cellulosic material is municipal solid waste. In anotheraspect, the cellulosic material is waste paper. In another aspect, thecellulosic material is pulp and paper mill residue.

In another aspect, the cellulosic material is corn stover. In anotheraspect, the cellulosic material is corn fiber. In another aspect, thecellulosic material is corn cob. In another aspect, the cellulosicmaterial is orange peel. In another aspect, the cellulosic material isrice straw. In another aspect, the cellulosic material is wheat straw.In another aspect, the cellulosic material is switch grass. In anotheraspect, the cellulosic material is miscanthus. In another aspect, thecellulosic material is bagasse.

In another aspect, the cellulosic material is microcrystallinecellulose. In another aspect, the cellulosic material is bacterialcellulose. In another aspect, the cellulosic material is algalcellulose. In another aspect, the cellulosic material is cotton linter.In another aspect, the cellulosic material is amorphous phosphoric-acidtreated cellulose. In another aspect, the cellulosic material is filterpaper.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

Pretreated corn stover: The term “PCS” or “Pretreated Corn Stover” isdefined herein as a cellulosic material derived from corn stover bytreatment with heat and dilute sulfuric acid.

Xylan-containing material: The term “xylan-containing material” isdefined herein as any material comprising a plant cell wallpolysaccharide containing a backbone of beta-(1-4)-linked xyloseresidues. Xylans of terrestrial plants are heteropolymers possessing abeta-(1-4)-D-xylopyranose backbone, which is branched by shortcarbohydrate chains. They comprise D-glucuronic acid or its 4-O-methylether, L-arabinose, and/or various oligosaccharides, composed ofD-xylose, L-arabinose, D- or L-galactose, and D-glucose. Xylan-typepolysaccharides can be divided into homoxylans and heteroxylans, whichinclude glucuronoxylans, (arabino)glucuronoxylans,(glucurono)arabinoxylans, arabinoxylans, and complex heteroxylans. See,for example, Ebringerova et al., 2005, Adv. Polym. Sci. 186: 1-67.

In the methods of the present invention, any material containing xylanmay be used. In a preferred aspect, the xylan-containing material islignocellulose.

Isolated polypeptide: The term “isolated polypeptide” as used hereinrefers to a polypeptide that is isolated from a source. In a preferredaspect, the polypeptide is at least 1% pure, preferably at least 5%pure, more preferably at least 10% pure, more preferably at least 20%pure, more preferably at least 40% pure, more preferably at least 60%pure, even more preferably at least 80% pure, and most preferably atleast 90% pure, as determined by SDS-PAGE.

Substantially pure polypeptide: The term “substantially purepolypeptide” denotes herein a polypeptide preparation that contains atmost 10%, preferably at most 8%, more preferably at most 6%, morepreferably at most 5%, more preferably at most 4%, more preferably atmost 3%, even more preferably at most 2%, most preferably at most 1%,and even most preferably at most 0.5% by weight of other polypeptidematerial with which it is natively or recombinantly associated. It is,therefore, preferred that the substantially pure polypeptide is at least92% pure, preferably at least 94% pure, more preferably at least 95%pure, more preferably at least 96% pure, more preferably at least 97%pure, more preferably at least 98% pure, even more preferably at least99% pure, most preferably at least 99.5% pure, and even most preferably100% pure by weight of the total polypeptide material present in thepreparation. The polypeptides are preferably in a substantially pureform, i.e., that the polypeptide preparation is essentially free ofother polypeptide material with which it is natively or recombinantlyassociated. This can be accomplished, for example, by preparing thepolypeptide by well-known recombinant methods or by classicalpurification methods.

Mature polypeptide: The term “mature polypeptide” is defined herein as apolypeptide in its final form following translation and anypost-translational modifications, such as N-terminal processing,C-terminal truncation, glycosylation, phosphorylation, etc.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” is defined herein as a nucleotide sequence that encodes amature polypeptide.

Identity: The relatedness between two amino acid sequences or betweentwo nucleotide sequences is described by the parameter “identity”.

For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends in Genetics 16: 276-277), preferably version 3.0.0 or later. Theoptional parameters used are gap open penalty of 10, gap extensionpenalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the—nobrief option) is used as the percent identity andis calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of identity betweentwo deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the—nobrief option)is used as the percent identity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Homologous sequence: The term “homologous sequence” is defined herein asa predicted protein having an E value (or expectancy score) of less than0.001 in a tfasty search (Pearson, W. R., 1999, in BioinformaticsMethods and Protocols, S. Misener and S. A. Krawetz, ed., pp. 185-219)with a polypeptide of interest.

Polypeptide fragment: The term “polypeptide fragment” is defined hereinas a polypeptide having one or more (several) amino acids deleted fromthe amino and/or carboxyl terminus of a mature polypeptide or ahomologous sequence thereof.

Subsequence: The term “subsequence” is defined herein as a nucleotidesequence having one or more (several) nucleotides deleted from the 5′and/or 3′ end of a mature polypeptide coding sequence or a homologoussequence thereof.

Allelic variant: The term “allelic variant” denotes herein any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Isolated polynucleotide: The term “isolated polynucleotide” as usedherein refers to a polynucleotide that is isolated from a source. In apreferred aspect, the polynucleotide is at least 1% pure, preferably atleast 5% pure, more preferably at least 10% pure, more preferably atleast 20% pure, more preferably at least 40% pure, more preferably atleast 60% pure, even more preferably at least 80% pure, and mostpreferably at least 90% pure, as determined by agarose electrophoresis.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” as used herein refers to a polynucleotide preparationfree of other extraneous or unwanted nucleotides and in a form suitablefor use within genetically engineered protein production systems. Thus,a substantially pure polynucleotide contains at most 10%, preferably atmost 8%, more preferably at most 6%, more preferably at most 5%, morepreferably at most 4%, more preferably at most 3%, even more preferablyat most 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polynucleotide material with which it isnatively or recombinantly associated. A substantially purepolynucleotide may, however, include naturally occurring 5′ and 3′untranslated regions, such as promoters and terminators. It is preferredthat the substantially pure polynucleotide is at least 90% pure,preferably at least 92% pure, more preferably at least 94% pure, morepreferably at least 95% pure, more preferably at least 96% pure, morepreferably at least 97% pure, even more preferably at least 98% pure,most preferably at least 99% pure, and even most preferably at least99.5% pure by weight. The polynucleotides are preferably in asubstantially pure form, i.e., that the polynucleotide preparation isessentially free of other polynucleotide material with which it isnatively or recombinantly associated. The polynucleotides may be ofgenomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinationsthereof.

Coding sequence: When used herein the term “coding sequence” means anucleotide sequence, which directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG and endswith a stop codon such as TAA, TAG, and TGA. The coding sequence may bea DNA, cDNA, synthetic, or recombinant nucleotide sequence.

cDNA: The term “cDNA” is defined herein as a DNA molecule that can beprepared by reverse transcription from a mature, spliced, mRNA moleculeobtained from a eukaryotic cell. cDNA lacks intron sequences that may bepresent in the corresponding genomic DNA. The initial, primary RNAtranscript is a precursor to mRNA that is processed through a series ofsteps before appearing as mature spliced mRNA. These steps include theremoval of intron sequences by a process called splicing. cDNA derivedfrom mRNA lacks, therefore, any intron sequences.

Nucleic acid construct: The term “nucleic acid construct” as used hereinrefers to a nucleic acid molecule, either single- or double-stranded,which is isolated from a naturally occurring gene or which is modifiedto contain segments of nucleic acids in a manner that would nototherwise exist in nature or which is synthetic. The term nucleic acidconstruct is synonymous with the term “expression cassette” when thenucleic acid construct contains the control sequences required forexpression of a coding sequence.

Control sequences: The term “control sequences” is defined herein toinclude all components necessary for the expression of a polynucleotideencoding a polypeptide. Each control sequence may be native or foreignto the nucleotide sequence encoding the polypeptide or native or foreignto each other. Such control sequences include, but are not limited to, aleader, polyadenylation sequence, propeptide sequence, promoter, signalpeptide sequence, and transcription terminator. At a minimum, thecontrol sequences include a promoter, and transcriptional andtranslational stop signals. The control sequences may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleotide sequence encoding a polypeptide.

Operably linked: The term “operably linked” denotes herein aconfiguration in which a control sequence is placed at an appropriateposition relative to the coding sequence of the polynucleotide sequencesuch that the control sequence directs the expression of the codingsequence of a polypeptide.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” is defined herein as alinear or circular DNA molecule that comprises a polynucleotide encodinga polypeptide and is operably linked to additional nucleotides thatprovide for its expression.

Host cell: The term “host cell”, as used herein, includes any cell typethat is susceptible to transformation, transfection, transduction, andthe like with a nucleic acid construct or expression vector comprising apolynucleotide of the present invention.

Modification: The term “modification” means herein any chemicalmodification of a polypeptide, as well as genetic manipulation of theDNA encoding the polypeptide. The modification can be a substitution, adeletion and/or an insertion of one or more (several) amino acids aswell as replacements of one or more (several) amino acid side chains.

Artificial variant: When used herein, the term “artificial variant”means a polypeptide produced by an organism expressing a modifiedpolynucleotide sequence encoding a polypeptide variant. The modifiednucleotide sequence is obtained through human intervention bymodification of the polynucleotide sequence.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for pretreating a cellulosicmaterial, comprising: pretreating the cellulosic material underanaerobic conditions. In one aspect, the method further comprisesrecovering the pretreated cellulosic material.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition under anaerobic conditions. In oneaspect, the method further comprises recovering the degraded orconverted cellulosic material.

The present invention also relates to methods for producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition under anaerobic conditions; (b)fermenting the saccharified cellulosic material with one or morefermenting microorganisms to produce the fermentation product; and (c)recovering the fermentation product from the fermentation.

The present invention further relates to methods of fermenting acellulosic material, comprising: fermenting the cellulosic material withone or more fermenting microorganisms, wherein the cellulosic materialis saccharified with an enzyme composition under anaerobic conditions.In one aspect, the fermenting of the cellulosic material produces afermentation product. In another aspect, the method further comprisesrecovering the fermentation product from the fermentation.

In the methods described above, pretreating or hydrolyzing thecellulosic material under anaerobic conditions improves the extentand/or rate of hydrolysis or conversion of cellulose to soluble,fermentable sugars by a cellulolytic enzyme composition.

In practicing the methods of the present invention, any anaerobic systemknown in the art can be used. Anaerobic reactors are widely used invarious industries, including wastewater treatment, industrial/municipalwaste digester, and biogas generation (see, for example,Castro-Gonzalez, A., Enriquez, M., Dura, C., 2001, Design, construction,and starting-up of an anaerobic reactor for the stabilisation, handling,and disposal of excess biological sludge generated in a wastewatertreatment plant, Anaerobe 7: 143-149; Melidis, P., Georgiou, D.,Aivasidis, A., 2003, Scale-up and design optimization of anaerobicimmobilized cell reactors for wastewater treatment, Chemical Engineeringand Processing 42: 897-908). In general, the system may include, but isnot limited to, one or more valved inlets to feed biomass feedstock,pretreatment agents (e.g., acid), inert (non-oxygen) gas, water,post-pretreatment agents (e.g., acid-neutralizing base), etc., one ormore valved outlets to dispense pretreated biomass, spent liquor,reacted/generated gas, etc., one or more mechanical stirring devices formixing, one or more monitors for pH, volume, gas, and temperaturecontrol, assembled in an enclosed assembly in which any contact withatmospheric oxygen is controlled/limited.

Anaerobicity may be achieved by air limitation, O₂ sequestration,bubbling/blanketing with an inert gas such as N₂, CO₂, Ar, etc., or acombination thereof. One preferred method is to fully fill a reactorwith degassed biomass suspension liquor with no/minimal head space.Another preferred way is to bubble and blanket a biomass hydrolysissuspension with CO₂, collected and delivered from a sugar-to-ethanolfermentation reactor where the gas is a by-product. CO₂ collection isused in several industries, such as beer manufacturing (for reference,see for example, Redford, S G (1997) Multi-stage column continuousfermentation system, U.S. Pat. No. 5,925,563; Coors Shenandoah: PeakPerformance, Beverage World, Jun. 15, 2008). A generic procedure can,but is not limited to, comprise a gas outlet mounted at the top of asugar-to-ethanol fermentor (to collect CO₂), an enclosed pipe (totransport CO₂) with or without a pressurizing pump, and aninjection/feeding inlet mounted to a biomass hydrolysis reactor (to feedCO₂).

The enzyme composition can be in the form of a crude fermentation brothwith or without the cells removed or in the form of a semi-purified orpurified enzyme preparation or the composition can comprise a host cellas a source of the enzyme composition in a fermentation process with thebiomass.

The methods of the present invention can be used to saccharify acellulosic material to fermentable sugars and convert the fermentablesugars to many useful substances, e.g., chemicals and fuels. Theproduction of a desired fermentation product from cellulosic materialtypically involves pretreatment, enzymatic hydrolysis(saccharification), and fermentation.

The treatment of cellulosic material according to the present inventioncan be accomplished using processes conventional in the art. Moreover,the methods of the present invention can be implemented using anyconventional biomass processing apparatus configured to operate inaccordance with the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and cofermentation (SSCF); hybridhydrolysis and fermentation (HHF); SHCF (separate hydrolysis andco-fermentation), HHCF (hybrid hydrolysis and fermentation), and directmicrobial conversion (DMC). SHF uses separate process steps to firstenzymatically hydrolyze lignocellulose to fermentable sugars, e.g.,glucose, cellobiose, cellotriose, and pentose sugars, and then fermentthe fermentable sugars to ethanol. In SSF, the enzymatic hydrolysis oflignocellulose and the fermentation of sugars to ethanol are combined inone step (Philippidis, G. P., 1996, Cellulose bioconversion technology,in Handbook on Bioethanol: Production and Utilization, Wyman, C. E.,ed., Taylor & Francis, Washington, D.C., 179-212). SSCF involves thecofermentation of multiple sugars (Sheehan, J., and Himmel, M., 1999,Enzymes, energy and the environment: A strategic perspective on the U.S.Department of Energy's research and development activities forbioethanol, Biotechnol. Prog. 15: 817-827). HHF involves a separatehydrolysis separate step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, lignocellulose hydrolysis, and fermentation) in oneor more steps where the same organism is used to produce the enzymes forconversion of the lignocellulose to fermentable sugars and to convertthe fermentable sugars into a final product (Lynd, L. R., Weimer, P. J.,van Zyl, W. H., and Pretorius, I. S., 2002, Microbial celluloseutilization: Fundamentals and biotechnology, Microbiol. Mol. Biol.Reviews 66: 506-577). It is understood herein that any method known inthe art comprising pretreatment, enzymatic hydrolysis(saccharification), fermentation, or a combination thereof can be usedin the practicing the methods of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (Fernandade Castilhos Corazza, Flávio Faria de Moraes, Gisella Maria Zanin andIvo Neitzel, 2003, Optimal control in fed-batch reactor for thecellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov,A. V., and Sinitsyn, A. P., 1985, Kinetics of the enzymatic hydrolysisof cellulose: 1. A mathematical model for a batch reactor process, Enz.Microb. Technol. 7: 346-352), an attrition reactor (Ryu, S. K., and Lee,J. M., 1983, Bioconversion of waste cellulose by using an attritionbioreactor, Biotechnol. Bioeng. 25: 53-65), or a reactor with intensivestirring induced by an electromagnetic field (Gusakov, A. V., Sinitsyn,A. P., Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., 1996,Enhancement of enzymatic cellulose hydrolysis using a novel type ofbioreactor with intensive stirring induced by electromagnetic field,Appl. Biochem. Biotechnol. 56: 141-153). Additional reactor typesinclude: Fluidized bed, upflow blanket, immobilized, and extruder typereactors for hydrolysis and/or fermentation.

Pretreatment. In practicing the methods of the present invention, anypretreatment process known in the art can be used to disrupt plant cellwall components of cellulosic material (Chandra et al., 2007, Substratepretreatment: The key to effective enzymatic hydrolysis oflignocellulosics? Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbe andZacchi, 2007, Pretreatment of lignocellulosic materials for efficientbioethanol production, Adv. Biochem. Engin./Biotechnol. 108: 41-65;Hendriks and Zeeman, 2009, Pretreatments to enhance the digestibility oflignocellulosic biomass, Bioresource Technol. 100: 10-18; Mosier et al.,2005, Features of promising technologies for pretreatment oflignocellulosic biomass, Bioresource Technol. 96: 673-686; Taherzadehand Karimi, 2008, Pretreatment of lignocellulosic wastes to improveethanol and biogas production: A review, Int. J. of Mol. Sci. 9:1621-1651; Yang and Wyman, 2008, Pretreatment: the key to unlockinglow-cost cellulosic ethanol, Biofuels Bioproducts andBiorefining-Biofpr. 2: 26-40).

The cellulosic material can also be subjected to particle sizereduction, pre-soaking, wetting, washing, or conditioning prior topretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ammonia percolation, ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O, ozone, and gamma irradiationpretreatments.

The cellulosic material can be pretreated before hydrolysis and/orfermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In most cases thepretreatment step itself results in some conversion of biomass tofermentable sugars (even in absence of enzymes).

Steam Pretreatment. In steam pretreatment, cellulosic material is heatedto disrupt the plant cell wall components, including lignin,hemicellulose, and cellulose to make the cellulose and other fractions,e.g., hemicellulose, accessible to enzymes. Cellulosic material ispassed to or through a reaction vessel where steam is injected toincrease the temperature to the required temperature and pressure and isretained therein for the desired reaction time. Steam pretreatment ispreferably done at 140-230° C., more preferably 160-200° C., and mostpreferably 170-190° C., where the optimal temperature range depends onany addition of a chemical catalyst. Residence time for the steampretreatment is preferably 1-15 minutes, more preferably 3-12 minutes,and most preferably 4-10 minutes, where the optimal residence timedepends on temperature range and any addition of a chemical catalyst.Steam pretreatment allows for relatively high solids loadings, so thatcellulosic material is generally only moist during the pretreatment. Thesteam pretreatment is often combined with an explosive discharge of thematerial after the pretreatment, which is known as steam explosion, thatis, rapid flashing to atmospheric pressure and turbulent flow of thematerial to increase the accessible surface area by fragmentation (Duffand Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi,2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. Patent ApplicationNo. 20020164730). During steam pretreatment, hemicellulose acetyl groupsare cleaved and the resulting acid autocatalyzes partial hydrolysis ofthe hemicellulose to monosaccharides and oligosaccharides. Lignin isremoved to only a limited extent.

A catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 3% w/w) is often addedprior to steam pretreatment, which decreases the time and temperature,increases the recovery, and improves enzymatic hydrolysis (Ballesteroset al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al.,2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006,Enzyme Microb. Technol. 39: 756-762).

Chemical Pretreatment: The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Examples of suitable chemicalpretreatment processes include, for example, dilute acid pretreatment,lime pretreatment, wet oxidation, ammonia fiber/freeze explosion (AFEX),ammonia percolation (APR), and organosolv pretreatments.

In dilute acid pretreatment, cellulosic material is mixed with diluteacid, typically H₂SO₄, and water to form a slurry, heated by steam tothe desired temperature, and after a residence time flashed toatmospheric pressure. The dilute acid pretreatment can be performed witha number of reactor designs, e.g., plug-flow reactors, counter-currentreactors, or continuous counter-current shrinking bed reactors (Duff andMurray, 1996, supra; Schell et al., 2004, Bioresource Technol. 91:179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to, limepretreatment, wet oxidation, ammonia percolation (APR), and ammoniafiber/freeze explosion (AFEX).

Lime pretreatment is performed with calcium carbonate, sodium hydroxide,or ammonia at low temperatures of 85-150° C. and residence times from 1hour to several days (Wyman et al., 2005, Bioresource Technol. 96:1959-1966; Mosier et al., 2005, Bioresource Technol. 96: 673-686). WO2006/110891, WO 2006/11899, WO 2006/11900, and WO 2006/110901 disclosepretreatment methods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technol. 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed at preferably 1-40% drymatter, more preferably 2-30% dry matter, and most preferably 5-20% drymatter, and often the initial pH is increased by the addition of alkalisuch as sodium carbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion), can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber explosion (AFEX) involves treating cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-100°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol.121: 1133-1141; Teymouri et al., 2005, Bioresource Technol. 96:2014-2018). AFEX pretreatment results in the depolymerization ofcellulose and partial hydrolysis of hemicellulose. Lignin-carbohydratecomplexes are cleaved.

Organosolv pretreatment delignifies cellulosic material by extractionusing aqueous ethanol (40-60% ethanol) at 160-200° C. for 30-60 minutes(Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan et al., 2006,Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl. Biochem.Biotechnol. 121: 219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of hemicellulose isremoved.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. and Biotechnol. Vol. 105-108, p. 69-85, andMosier et al., 2005, Bioresource Technology 96: 673-686, and U.S.Published Application 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as anacid treatment, and more preferably as a continuous dilute and/or mildacid treatment. The acid is typically sulfuric acid, but other acids canalso be used, such as acetic acid, citric acid, nitric acid, phosphoricacid, tartaric acid, succinic acid, hydrogen chloride, or mixturesthereof. Mild acid treatment is conducted in the pH range of preferably1-5, more preferably 1-4, and most preferably 1-3. In one aspect, theacid concentration is in the range from preferably 0.01 to 20 wt % acid,more preferably 0.05 to 10 wt % acid, even more preferably 0.1 to 5 wt %acid, and most preferably 0.2 to 2.0 wt % acid. The acid is contactedwith cellulosic material and held at a temperature in the range ofpreferably 160-220° C., and more preferably 165-195° C., for periodsranging from seconds to minutes to, e.g., 1 second to 60 minutes.

In another aspect, pretreatment is carried out as an ammonia fiberexplosion step (AFEX pretreatment step).

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, cellulosic material is present during pretreatment inamounts preferably between 10-80 wt %, more preferably between 20-70 wt%, and most preferably between 30-60 wt %, such as around 50 wt %. Thepretreated cellulosic material can be unwashed or washed using anymethod known in the art, e.g., washed with water.

Mechanical Pretreatment: The term “mechanical pretreatment” refers tovarious types of grinding or milling (e.g., dry milling, wet milling, orvibratory ball milling).

Physical Pretreatment: The term “physical pretreatment” refers to anypretreatment that promotes the separation and/or release of cellulose,hemicellulose, and/or lignin from cellulosic material. For example,physical pretreatment can involve irradiation (e.g., microwaveirradiation), steaming/steam explosion, hydrothermolysis, andcombinations thereof.

Physical pretreatment can involve high pressure and/or high temperature(steam explosion). In one aspect, high pressure means pressure in therange of preferably about 300 to about 600 psi, more preferably about350 to about 550 psi, and most preferably about 400 to about 500 psi,such as around 450 psi. In another aspect, high temperature meanstemperatures in the range of about 100 to about 300° C., preferablyabout 140 to about 235° C. In a preferred aspect, mechanicalpretreatment is performed in a batch-process, steam gun hydrolyzersystem that uses high pressure and high temperature as defined above,e.g., a Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden.

Combined Physical and Chemical Pretreatment: Cellulosic material can bepretreated both physically and chemically. For instance, thepretreatment step can involve dilute or mild acid treatment and hightemperature and/or pressure treatment. The physical and chemicalpretreatments can be carried out sequentially or simultaneously, asdesired. A mechanical pretreatment can also be included.

Accordingly, in a preferred aspect, cellulosic material is subjected tomechanical, chemical, or physical pretreatment, or any combinationthereof, to promote the separation and/or release of cellulose,hemicellulose, and/or lignin.

Biological Pretreatment: The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from cellulosic material.Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms (see, for example, Hsu, T.-A., 1996,Pretreatment of biomass, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212; Ghosh and Singh, 1993, Physicochemical and biologicaltreatments for enzymatic/microbial conversion of cellulosic biomass,Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994, Pretreatinglignocellulosic biomass: a review, in Enzymatic Conversion of Biomassfor Fuels Production, Himmel, M. E., Baker, J. O., and Overend, R. P.,eds., ACS Symposium Series 566, American Chemical Society, Washington,D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T.,1999, Ethanol production from renewable resources, in Advances inBiochemical Engineering/Biotechnology, Scheper, T., ed., Springer-VerlagBerlin Heidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996,Fermentation of lignocellulosic hydrolysates for ethanol production,Enz. Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990,Production of ethanol from lignocellulosic materials: State of the art,Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification. In the hydrolysis step, also known assaccharification, the pretreated cellulosic material is hydrolyzed tobreak down cellulose and alternatively also hemicellulose to fermentablesugars, such as glucose, cellobiose, xylose, xylulose, arabinose,mannose, galactose, and/or soluble oligosaccharides. The hydrolysis isperformed enzymatically by an enzyme composition described herein. Theenzymes of the compositions can also be added sequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In a preferred aspect, hydrolysis is performed underconditions suitable for the activity of the enzyme(s), i.e., optimal forthe enzyme(s). The hydrolysis can be carried out as a fed batch orcontinuous process where the pretreated cellulosic material (substrate)is fed gradually to, for example, an enzyme containing hydrolysissolution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 96 hours, more preferably about 16 to about 72 hours,and most preferably about 24 to about 48 hours. The temperature is inthe range of preferably about 25° C. to about 70° C., more preferablyabout 30° C. to about 65° C., and more preferably about 40° C. to 60°C., in particular about 50° C. The pH is in the range of preferablyabout 3 to about 8, more preferably about 3.5 to about 7, and mostpreferably about 4 to about 6, in particular about pH 5. The dry solidscontent is in the range of preferably about 5 to about 50 wt %, morepreferably about 10 to about 40 wt %, and most preferably about 20 toabout 30 wt %.

The optimum amounts of the enzymes depend on several factors including,but not limited to, the mixture of component cellulolytic enzymes, thecellulosic substrate, the concentration of cellulosic substrate, thepretreatment(s) of the cellulosic substrate, temperature, time, pH, andinclusion of fermenting organism (e.g., yeast for SimultaneousSaccharification and Fermentation).

In a preferred aspect, an effective amount of cellulolytic enzyme(s) tocellulosic material is about 0.5 to about 50 mg, preferably at about 0.5to about 40 mg, more preferably at about 0.5 to about 25 mg, morepreferably at about 0.75 to about 20 mg, more preferably at about 0.75to about 15 mg, even more preferably at about 0.5 to about 10 mg, andmost preferably at about 2.5 to about 10 mg per g of cellulosicmaterial.

In another preferred aspect, an effective amount of polypeptide(s)having cellulolytic enhancing activity to cellulosic material is about0.01 to about 50.0 mg, preferably about 0.01 to about 40 mg, morepreferably about 0.01 to about 30 mg, more preferably about 0.01 toabout 20 mg, more preferably about 0.01 to about 10 mg, more preferablyabout 0.01 to about 5 mg, more preferably at about 0.025 to about 1.5mg, more preferably at about 0.05 to about 1.25 mg, more preferably atabout 0.075 to about 1.25 mg, more preferably at about 0.1 to about 1.25mg, even more preferably at about 0.15 to about 1.25 mg, and mostpreferably at about 0.25 to about 1.0 mg per g of cellulosic material.

In another preferred aspect, an effective amount of polypeptide(s)having cellulolytic enhancing activity to cellulolytic protein(s) isabout 0.005 to about 1.0 g, preferably at about 0.01 to about 1.0 g,more preferably at about 0.15 to about 0.75 g, more preferably at about0.15 to about 0.5 g, more preferably at about 0.1 to about 0.5 g, evenmore preferably at about 0.1 to about 0.5 g, and most preferably atabout 0.05 to about 0.2 g per g of cellulolytic protein(s).

Fermentation. The fermentable sugars obtained from the pretreated andhydrolyzed cellulosic material can be fermented by one or more (several)fermenting microorganisms capable of fermenting the sugars directly orindirectly into a desired fermentation product. “Fermentation” or“fermentation process” refers to any fermentation process or any processcomprising a fermentation step. Fermentation processes also includefermentation processes used in the consumable alcohol industry (e.g.,beer and wine), dairy industry (e.g., fermented dairy products), leatherindustry, and tobacco industry. The fermentation conditions depend onthe desired fermentation product and fermenting organism and can easilybe determined by one skilled in the art.

In the fermentation step, sugars, released from cellulosic material as aresult of the pretreatment and enzymatic hydrolysis steps, are fermentedto a product, e.g., ethanol, by a fermenting organism, such as yeast.Hydrolysis (saccharification) and fermentation can be separate orsimultaneous, as described herein.

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on the desired fermentation product, i.e., thesubstance to be obtained from the fermentation, and the processemployed, as is well known in the art.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be C₆ and/or C₅ fermenting organisms, or a combinationthereof. Both C₆ and C₅ fermenting organisms are well known in the art.Suitable fermenting microorganisms are able to ferment, i.e., convert,sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose,galactose, or oligosaccharides, directly or indirectly into the desiredfermentation product.

Examples of bacterial and fungal fermenting organisms producing ethanolare described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69:627-642.

Examples of fermenting microorganisms that can ferment C₆ sugars includebacterial and fungal organisms, such as yeast. Preferred yeast includesstrains of the Saccharomyces spp., preferably Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment C₅ sugars includebacterial and fungal organisms, such as yeast. Preferred C₅ fermentingyeast include strains of Pichia, preferably Pichia stipitis, such asPichia stipitis CBS 5773; strains of Candida, preferably Candidaboidinii, Candida brassicae, Candida sheatae, Candida diddensii, Candidapseudotropicalis, or Candida utilis.

Other fermenting organisms include strains of Zymomonas, such asZymomonas mobilis; Hansenula, such as Hansenula anomala; Kluyveromyces,such as K. fragilis; Schizosaccharomyces, such as S. pombe; and E. coli,especially E. coli strains that have been genetically modified toimprove the yield of ethanol.

In a preferred aspect, the yeast is a Saccharomyces spp. In a morepreferred aspect, the yeast is Saccharomyces cerevisiae. In another morepreferred aspect, the yeast is Saccharomyces distaticus. In another morepreferred aspect, the yeast is Saccharomyces uvarum. In anotherpreferred aspect, the yeast is a Kluyveromyces. In another morepreferred aspect, the yeast is Kluyveromyces marxianus. In another morepreferred aspect, the yeast is Kluyveromyces fragilis. In anotherpreferred aspect, the yeast is a Candida. In another more preferredaspect, the yeast is Candida boidinii. In another more preferred aspect,the yeast is Candida brassicae. In another more preferred aspect, theyeast is Candida diddensii. In another more preferred aspect, the yeastis Candida pseudotropicalis. In another more preferred aspect, the yeastis Candida utilis. In another preferred aspect, the yeast is aClavispora. In another more preferred aspect, the yeast is Clavisporalusitaniae. In another more preferred aspect, the yeast is Clavisporaopuntiae. In another preferred aspect, the yeast is a Pachysolen. Inanother more preferred aspect, the yeast is Pachysolen tannophilus. Inanother preferred aspect, the yeast is a Pichia. In another morepreferred aspect, the yeast is a Pichia stipitis. In another preferredaspect, the yeast is a Bretannomyces. In another more preferred aspect,the yeast is Bretannomyces clausenii (Philippidis, G. P., 1996,Cellulose bioconversion technology, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212).

Bacteria that can efficiently ferment hexose and pentose to ethanolinclude, for example, Zymomonas mobilis and Clostridium thermocellum(Philippidis, 1996, supra).

In a preferred aspect, the bacterium is a Zymomonas. In a more preferredaspect, the bacterium is Zymomonas mobilis. In another preferred aspect,the bacterium is a Clostridium. In another more preferred aspect, thebacterium is Clostridium thermocellum.

Commercially available yeast suitable for ethanol production includes,e.g., ETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI™(available from Fleischmann's Yeast, USA), SUPERSTART™ and THERMOSACC™fresh yeast (available from Ethanol Technology, WI, USA), BIOFERM™ AFTand XR (available from NABC—North American Bioproducts Corporation, GA,USA), GERT STRAND™ (available from Gert Strand AB, Sweden), and FERMIOL™(available from DSM Specialties).

In a preferred aspect, the fermenting microorganism has been geneticallymodified to provide the ability to ferment pentose sugars, such asxylose utilizing, arabinose utilizing, and xylose and arabinoseco-utilizing microorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (cofermentation) (Chen and Ho, 1993, Cloning andimproving the expression of Pichia stipitis xylose reductase gene inSaccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Hoet al., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation bySaccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783;Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiaestrains overexpressing the TKL1 and TAL1 genes encoding the pentosephosphate pathway enzymes transketolase and transaldolase, Appl.Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimalmetabolic engineering of Saccharomyces cerevisiae for efficientanaerobic xylose fermentation: a proof of principle, FEMS Yeast Research4: 655-664; Beall et al., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering ofbacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhanget al., 1995, Metabolic engineering of a pentose metabolism pathway inethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al.,1996, Development of an arabinose-fermenting Zymomonas mobilis strain bymetabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470;WO 2003/062430, xylose isomerase).

In a preferred aspect, the genetically modified fermenting microorganismis Saccharomyces cerevisiae. In another preferred aspect, thegenetically modified fermenting microorganism is Zymomonas mobilis. Inanother preferred aspect, the genetically modified fermentingmicroorganism is Escherichia coli. In another preferred aspect, thegenetically modified fermenting microorganism is Klebsiella oxytoca. Inanother preferred aspect, the genetically modified fermentingmicroorganism is Kluyveromyces sp.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedlignocellulose or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, such as about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., inparticular about 32° C. or 50° C., and at about pH 3 to about pH 8, suchas around pH 4-5, 6, or 7.

In a preferred aspect, the yeast and/or another microorganism is appliedto the degraded cellulosic material and the fermentation is performedfor about 12 to about 96 hours, such as typically 24-60 hours. In apreferred aspect, the temperature is preferably between about 20° C. toabout 60° C., more preferably about 25° C. to about 50° C., and mostpreferably about 32° C. to about 50° C., in particular about 32° C. or50° C., and the pH is generally from about pH 3 to about pH 7,preferably around pH 4-7. However, some fermenting organisms, e.g.,bacteria, have higher fermentation temperature optima. Yeast or anothermicroorganism is preferably applied in amounts of approximately 10⁵ to10¹², preferably from approximately 10⁷ to 10¹⁰, especiallyapproximately 2×10⁸ viable cell count per ml of fermentation broth.Further guidance in respect of using yeast for fermentation can be foundin, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P. Lyons and D.R. Kelsall, Nottingham University Press, United Kingdom 1999), which ishereby incorporated by reference.

For ethanol production, following the fermentation the fermented slurryis distilled to extract the ethanol. The ethanol obtained according tothe methods of the invention can be used as, e.g., fuel ethanol,drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can Supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation products: A fermentation product can be any substancederived from the fermentation. The fermentation product can be, withoutlimitation, an alcohol (e.g., arabinitol, butanol, ethanol, glycerol,methanol, 1,3-propanediol, sorbitol, and xylitol); an organic acid(e.g., acetic acid, acetonic acid, adipic acid, ascorbic acid, citricacid, 2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaricacid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionicacid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid,oxaloacetic acid, propionic acid, succinic acid, and xylonic acid); aketone (e.g., acetone); an amino acid (e.g., aspartic acid, glutamicacid, glycine, lysine, serine, and threonine); and a gas (e.g., methane,hydrogen (H₂), carbon dioxide (CO₂), and carbon monoxide (CO)). Thefermentation product can also be protein as a high value product.

In a preferred aspect, the fermentation product is an alcohol. It willbe understood that the term “alcohol” encompasses a substance thatcontains one or more hydroxyl moieties. In a more preferred aspect, thealcohol is arabinitol. In another more preferred aspect, the alcohol isbutanol. In another more preferred aspect, the alcohol is ethanol. Inanother more preferred aspect, the alcohol is glycerol. In another morepreferred aspect, the alcohol is methanol. In another more preferredaspect, the alcohol is 1,3-propanediol. In another more preferredaspect, the alcohol is sorbitol. In another more preferred aspect, thealcohol is xylitol. See, for example, Gong, C. S., Cao, N. J., Du, J.,and Tsao, G. T., 1999, Ethanol production from renewable resources, inAdvances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira, M.M., and Jonas, R., 2002, The biotechnological production of sorbitol,Appl. Microbiol. Biotechnol. 59: 400-408; Nigam, P., and Singh, D.,1995, Processes for fermentative production of xylitol—a sugarsubstitute, Process Biochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi,N. and Blaschek, H. P., 2003, Production of acetone, butanol and ethanolby Clostridium beijerinckii BA101 and in situ recovery by gas stripping,World Journal of Microbiology and Biotechnology 19 (6): 595-603.

In another preferred aspect, the fermentation product is an organicacid. In another more preferred aspect, the organic acid is acetic acid.In another more preferred aspect, the organic acid is acetonic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid. Inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid. Inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid. Inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediatedextractive fermentation for lactic acid production from cellulosicbiomass, Appl. Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the fermentation product is a ketone. Itwill be understood that the term “ketone” encompasses a substance thatcontains one or more ketone moieties. In another more preferred aspect,the ketone is acetone. See, for example, Qureshi and Blaschek, 2003,supra.

In another preferred aspect, the fermentation product is an amino acid.In another more preferred aspect, the organic acid is aspartic acid. Inanother more preferred aspect, the amino acid is glutamic acid. Inanother more preferred aspect, the amino acid is glycine. In anothermore preferred aspect, the amino acid is lysine. In another morepreferred aspect, the amino acid is serine. In another more preferredaspect, the amino acid is threonine. See, for example, Richard, A., andMargaritis, A., 2004, Empirical modeling of batch fermentation kineticsfor poly(glutamic acid) production and other microbial biopolymers,Biotechnology and Bioengineering 87 (4): 501-515.

In another preferred aspect, the fermentation product is a gas. Inanother more preferred aspect, the gas is methane. In another morepreferred aspect, the gas is H₂. In another more preferred aspect, thegas is CO₂. In another more preferred aspect, the gas is CO. See, forexample, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies onhydrogen production by continuous culture system of hydrogen-producinganaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; andGunaseelan V. N. in Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114,1997, Anaerobic digestion of biomass for methane production: A review.

Recovery. The fermentation product(s) can be optionally recovered fromthe fermentation medium using any method known in the art including, butnot limited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic material and purified byconventional methods of distillation. Ethanol with a purity of up toabout 96 vol. % can be obtained, which can be used as, for example, fuelethanol, drinking ethanol, i.e., potable neutral spirits, or industrialethanol.

Polypeptides Having Cellulolytic Enhancing Activity and PolynucleotidesThereof

In the methods of the present invention, any polypeptide havingcellulolytic enhancing activity can be used.

In a first aspect, the polypeptide having cellulolytic enhancingactivity comprises the following motifs:

[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ] (SEQ ID NO: 71 or SEQID NO: 72) and [FW]-[TF]-K-[AIV],

wherein X is any amino acid, X(4,5) is any amino acid at 4 or 5contiguous positions, and X(4) is any amino acid at 4 contiguouspositions.

The polypeptide comprising the above-noted motifs may further comprise:

H-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 73 or SEQ ID NO: 74),

[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 75), or

H-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 76 or SEQ ID NO: 77) and[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 78),

wherein X is any amino acid, X(1,2) is any amino acid at 1 position or 2contiguous positions, X(3) is any amino acid at 3 contiguous positions,and X(2) is any amino acid at 2 contiguous positions. In the abovemotifs, the accepted IUPAC single letter amino acid abbreviation isemployed.

In a preferred aspect, the polypeptide having cellulolytic enhancingactivity further comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 73or SEQ ID NO: 74). In another preferred aspect, the isolated polypeptidehaving cellulolytic enhancing activity further comprises[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 75). In anotherpreferred aspect, the polypeptide having cellulolytic enhancing activityfurther comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 76 or SEQID NO: 77) and [EQ]X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 78).

In a second aspect, the polypeptide having cellulolytic enhancingactivity comprises the following motif:

[ILMV]-P-x(4,5)-G-x-Y-[ILMV]-x-R-x-[EQ]-x(3)-A-[HNQ] (SEQ ID NO: 79 orSEQ ID NO: 80),

wherein x is any amino acid, x(4,5) is any amino acid at 4 or 5contiguous positions, and x(3) is any amino acid at 3 contiguouspositions. In the above motif, the accepted IUPAC single letter aminoacid abbreviation is employed.

In a third aspect, the polypeptide having cellulolytic enhancingactivity comprises an amino acid sequence that has a degree of identityto the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO:16 of preferably at least 60%, more preferably at least 65%, morepreferably at least 70%, more preferably at least 75%, more preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, most preferably at least 95%, and even most preferably atleast 96%, at least 97%, at least 98%, or at least 99% (hereinafter“homologous polypeptides”). In a preferred aspect, the maturepolypeptide sequence is amino acids 20 to 326 of SEQ ID NO: 2, aminoacids 18 to 239 of SEQ ID NO: 4, amino acids 20 to 258 of SEQ ID NO: 6,amino acids 19 to 226 of SEQ ID NO: 8, amino acids 20 to 304 of SEQ IDNO: 10, amino acids 16 to 317 of SEQ ID NO: 12, amino acids 23 to 250 ofSEQ ID NO: 14, or amino acids 20 to 249 of SEQ ID NO: 16.

A polypeptide having cellulolytic enhancing activity preferablycomprises the amino acid sequence of SEQ ID NO: 2 or an allelic variantthereof; or a fragment thereof that has cellulolytic enhancing activity.In a preferred aspect, the polypeptide comprises the amino acid sequenceof SEQ ID NO: 2. In another preferred aspect, the polypeptide comprisesthe mature polypeptide of SEQ ID NO: 2. In another preferred aspect, thepolypeptide comprises amino acids 20 to 326 of SEQ ID NO: 2, or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptidecomprises amino acids 20 to 326 of SEQ ID NO: 2. In another preferredaspect, the polypeptide consists of the amino acid sequence of SEQ IDNO: 2 or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 2. Inanother preferred aspect, the polypeptide consists of the maturepolypeptide of SEQ ID NO: 2. In another preferred aspect, thepolypeptide consists of amino acids 20 to 326 of SEQ ID NO: 2 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptideconsists of amino acids 20 to 326 of SEQ ID NO: 2.

A polypeptide having cellulolytic enhancing activity preferablycomprises the amino acid sequence of SEQ ID NO: 4 or an allelic variantthereof; or a fragment thereof that has cellulolytic enhancing activity.In a preferred aspect, the polypeptide comprises the amino acid sequenceof SEQ ID NO: 4. In another preferred aspect, the polypeptide comprisesthe mature polypeptide of SEQ ID NO: 4. In another preferred aspect, thepolypeptide comprises amino acids 18 to 239 of SEQ ID NO: 4, or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptidecomprises amino acids 18 to 239 of SEQ ID NO: 4. In another preferredaspect, the polypeptide consists of the amino acid sequence of SEQ IDNO: 4 or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 4. Inanother preferred aspect, the polypeptide consists of the maturepolypeptide of SEQ ID NO: 4. In another preferred aspect, thepolypeptide consists of amino acids 18 to 239 of SEQ ID NO: 4 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptideconsists of amino acids 18 to 239 of SEQ ID NO: 4.

A polypeptide having cellulolytic enhancing activity preferablycomprises the amino acid sequence of SEQ ID NO: 6 or an allelic variantthereof; or a fragment thereof that has cellulolytic enhancing activity.In a preferred aspect, the polypeptide comprises the amino acid sequenceof SEQ ID NO: 6. In another preferred aspect, the polypeptide comprisesthe mature polypeptide of SEQ ID NO: 6. In another preferred aspect, thepolypeptide comprises amino acids 20 to 258 of SEQ ID NO: 6, or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptidecomprises amino acids 20 to 258 of SEQ ID NO: 6. In another preferredaspect, the polypeptide consists of the amino acid sequence of SEQ IDNO: 6 or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 6. Inanother preferred aspect, the polypeptide consists of the maturepolypeptide of SEQ ID NO: 6. In another preferred aspect, thepolypeptide consists of amino acids 20 to 258 of SEQ ID NO: 6 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptideconsists of amino acids 20 to 258 of SEQ ID NO: 6.

A polypeptide having cellulolytic enhancing activity preferablycomprises the amino acid sequence of SEQ ID NO: 8 or an allelic variantthereof; or a fragment thereof that has cellulolytic enhancing activity.In a preferred aspect, the polypeptide comprises the amino acid sequenceof SEQ ID NO: 8. In another preferred aspect, the polypeptide comprisesthe mature polypeptide of SEQ ID NO: 8. In another preferred aspect, thepolypeptide comprises amino acids 19 to 226 of SEQ ID NO: 8, or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptidecomprises amino acids 19 to 226 of SEQ ID NO: 8. In another preferredaspect, the polypeptide consists of the amino acid sequence of SEQ IDNO: 8 or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 8. Inanother preferred aspect, the polypeptide consists of the maturepolypeptide of SEQ ID NO: 8. In another preferred aspect, thepolypeptide consists of amino acids 19 to 226 of SEQ ID NO: 8 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptideconsists of amino acids 19 to 226 of SEQ ID NO: 8.

A polypeptide having cellulolytic enhancing activity preferablycomprises the amino acid sequence of SEQ ID NO: 10 or an allelic variantthereof; or a fragment thereof that has cellulolytic enhancing activity.In a preferred aspect, the polypeptide comprises the amino acid sequenceof SEQ ID NO: 10. In another preferred aspect, the polypeptide comprisesthe mature polypeptide of SEQ ID NO: 10. In another preferred aspect,the polypeptide comprises amino acids 20 to 304 of SEQ ID NO: 10, or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptidecomprises amino acids 20 to 304 of SEQ ID NO: 10. In another preferredaspect, the polypeptide consists of the amino acid sequence of SEQ IDNO: 10 or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 10. Inanother preferred aspect, the polypeptide consists of the maturepolypeptide of SEQ ID NO: 10. In another preferred aspect, thepolypeptide consists of amino acids 20 to 304 of SEQ ID NO: 10 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptideconsists of amino acids 20 to 304 of SEQ ID NO: 10.

A polypeptide having cellulolytic enhancing activity preferablycomprises the amino acid sequence of SEQ ID NO: 12 or an allelic variantthereof; or a fragment thereof having cellulolytic enhancing activity.In a preferred aspect, the polypeptide comprises the amino acid sequenceof SEQ ID NO: 12. In another preferred aspect, the polypeptide comprisesthe mature polypeptide of SEQ ID NO: 12. In another preferred aspect,the polypeptide comprises amino acids 16 to 317 of SEQ ID NO: 12, or anallelic variant thereof; or a fragment thereof having cellulolyticenhancing activity. In another preferred aspect, the polypeptidecomprises amino acids 16 to 317 of SEQ ID NO: 12. In another preferredaspect, the polypeptide consists of the amino acid sequence of SEQ IDNO: 12 or an allelic variant thereof; or a fragment thereof havingcellulolytic enhancing activity. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 12. Inanother preferred aspect, the polypeptide consists of the maturepolypeptide of SEQ ID NO: 12. In another preferred aspect, thepolypeptide consists of amino acids 16 to 317 of SEQ ID NO: 12 or anallelic variant thereof; or a fragment thereof having cellulolyticenhancing activity. In another preferred aspect, the polypeptideconsists of amino acids 16 to 317 of SEQ ID NO: 12.

A polypeptide having cellulolytic enhancing activity preferablycomprises the amino acid sequence of SEQ ID NO: 14 or an allelic variantthereof; or a fragment thereof that has cellulolytic enhancing activity.In a preferred aspect, the polypeptide comprises the amino acid sequenceof SEQ ID NO: 14. In another preferred aspect, the polypeptide comprisesthe mature polypeptide of SEQ ID NO: 14. In another preferred aspect,the polypeptide comprises amino acids 23 to 250 of SEQ ID NO: 14, or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptidecomprises amino acids 23 to 250 of SEQ ID NO: 14. In another preferredaspect, the polypeptide consists of the amino acid sequence of SEQ IDNO: 14 or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 14. Inanother preferred aspect, the polypeptide consists of the maturepolypeptide of SEQ ID NO: 14. In another preferred aspect, thepolypeptide consists of amino acids 23 to 250 of SEQ ID NO: 14 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptideconsists of amino acids 23 to 250 of SEQ ID NO: 14.

A polypeptide having cellulolytic enhancing activity preferablycomprises the amino acid sequence of SEQ ID NO: 16 or an allelic variantthereof; or a fragment thereof that has cellulolytic enhancing activity.In a preferred aspect, the polypeptide comprises the amino acid sequenceof SEQ ID NO: 16. In another preferred aspect, the polypeptide comprisesthe mature polypeptide of SEQ ID NO: 16. In another preferred aspect,the polypeptide comprises amino acids 20 to 249 of SEQ ID NO: 16, or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptidecomprises amino acids 20 to 249 of SEQ ID NO: 16. In another preferredaspect, the polypeptide consists of the amino acid sequence of SEQ IDNO: 16 or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 16. Inanother preferred aspect, the polypeptide consists of the maturepolypeptide of SEQ ID NO: 16. In another preferred aspect, thepolypeptide consists of amino acids 20 to 249 of SEQ ID NO: 16 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptideconsists of amino acids 20 to 249 of SEQ ID NO: 16.

Preferably, a fragment of the mature polypeptide of SEQ ID NO: 2contains at least 277 amino acid residues, more preferably at least 287amino acid residues, and most preferably at least 297 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:4 contains at least 185 amino acid residues, more preferably at least195 amino acid residues, and most preferably at least 205 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:6 contains at least 200 amino acid residues, more preferably at least212 amino acid residues, and most preferably at least 224 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:8 contains at least 175 amino acid residues, more preferably at least185 amino acid residues, and most preferably at least 195 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:10 contains at least 240 amino acid residues, more preferably at least255 amino acid residues, and most preferably at least 270 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:12 contains at least 255 amino acid residues, more preferably at least270 amino acid residues, and most preferably at least 285 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:14 contains at least 175 amino acid residues, more preferably at least190 amino acid residues, and most preferably at least 205 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:16 contains at least 200 amino acid residues, more preferably at least210 amino acid residues, and most preferably at least 220 amino acidresidues.

Preferably, a subsequence of the mature polypeptide coding sequence ofSEQ ID NO: 1 contains at least 831 nucleotides, more preferably at least861 nucleotides, and most preferably at least 891 nucleotides.Preferably, a subsequence of the mature polypeptide coding sequence ofSEQ ID NO: 3 contains at least 555 nucleotides, more preferably at least585 nucleotides, and most preferably at least 615 nucleotides.Preferably, a subsequence of the mature polypeptide coding sequence ofSEQ ID NO: 5 contains at least 600 nucleotides, more preferably at least636 nucleotides, and most preferably at least 672 nucleotides.Preferably, a subsequence of the mature polypeptide coding sequence ofSEQ ID NO: 7 contains at least 525 nucleotides, more preferably at least555 nucleotides, and most preferably at least 585 nucleotides.Preferably, a subsequence of the mature polypeptide coding sequence ofSEQ ID NO: 9 contains at least 720 nucleotides, more preferably at least765 nucleotides, and most preferably at least 810 nucleotides.Preferably, a subsequence of the mature polypeptide coding sequence ofSEQ ID NO: 11 contains at least 765 nucleotides, more preferably atleast 810 nucleotides, and most preferably at least 855 nucleotidesPreferably, a subsequence of the mature polypeptide coding sequence ofnucleotides 67 to 796 of SEQ ID NO: 13 contains at least 525nucleotides, more preferably at least 570 nucleotides, and mostpreferably at least 615 nucleotides. Preferably, a subsequence of themature polypeptide coding sequence of SEQ ID NO: 15 contains at least600 nucleotides, more preferably at least 630 nucleotides, and mostpreferably at least 660 nucleotides.

In a fourth aspect, the polypeptide having cellulolytic enhancingactivity is encoded by a polynucleotide that hybridizes under at leastvery low stringency conditions, preferably at least low stringencyconditions, more preferably at least medium stringency conditions, morepreferably at least medium-high stringency conditions, even morepreferably at least high stringency conditions, and most preferably atleast very high stringency conditions with (i) the mature polypeptidecoding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15, (ii)the cDNA sequence contained in the mature polypeptide coding sequence ofSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 13, or thegenomic DNA sequence comprising the mature polypeptide coding sequenceof SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 15, (iii) asubsequence of (i) or (ii), or (iv) a full-length complementary strandof (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatus,1989, supra). A subsequence of the mature polypeptide coding sequence ofSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15 contains at least 100contiguous nucleotides or preferably at least 200 contiguousnucleotides. Moreover, the subsequence may encode a polypeptide fragmentthat has cellulolytic enhancing activity. In a preferred aspect, themature polypeptide coding sequence is nucleotides 388 to 1332 of SEQ IDNO: 1, nucleotides 98 to 821 of SEQ ID NO: 3, nucleotides 126 to 978 ofSEQ ID NO: 5, nucleotides 55 to 678 of SEQ ID NO: 7, nucleotides 58 to912 of SEQ ID NO: 9, nucleotides 46 to 951 of SEQ ID NO: 11, nucleotides67 to 796 of SEQ ID NO: 13, or nucleotides 77 to 766 of SEQ ID NO: 15.

The nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15,or a subsequence thereof; as well as the amino acid sequence of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ IDNO: 12, SEQ ID NO: 14, or SEQ ID NO: 16, or a fragment thereof, may beused to design a nucleic acid probe to identify and clone DNA encodingpolypeptides having cellulolytic enhancing activity from strains ofdifferent genera or species according to methods well known in the art.In particular, such probes can be used for hybridization with thegenomic or cDNA of the genus or species of interest, following standardSouthern blotting procedures, in order to identify and isolate thecorresponding gene therein. Such probes can be considerably shorter thanthe entire sequence, but should be at least 14, preferably at least 25,more preferably at least 35, and most preferably at least 70 nucleotidesin length. It is, however, preferred that the nucleic acid probe is atleast 100 nucleotides in length. For example, the nucleic acid probe maybe at least 200 nucleotides, preferably at least 300 nucleotides, morepreferably at least 400 nucleotides, or most preferably at least 500nucleotides in length. Even longer probes may be used, e.g., nucleicacid probes that are preferably at least 600 nucleotides, morepreferably at least 700 nucleotides, even more preferably at least 800nucleotides, or most preferably at least 900 nucleotides in length. BothDNA and RNA probes can be used. The probes are typically labeled fordetecting the corresponding gene (for example, with ³²P, ³H, ³⁵S,biotin, or avidin). Such probes are encompassed by the presentinvention.

A genomic DNA or cDNA library prepared from such other strains may,therefore, be screened for DNA that hybridizes with the probes describedabove and encodes a polypeptide having cellulolytic enhancing activity.Genomic or other DNA from such other strains may be separated by agaroseor polyacrylamide gel electrophoresis, or other separation techniques.DNA from the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA that is homologous with SEQ ID NO: 1,or a subsequence thereof, the carrier material is preferably used in aSouthern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled nucleic acid probecorresponding to the mature polypeptide coding sequence of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, or SEQ ID NO: 15 the cDNA sequence contained in themature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, or SEQ ID NO: 13, or the genomic DNA sequence comprising themature polypeptide coding sequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 11, or SEQ ID NO: 15, its full-length complementary strand, or asubsequence thereof, under very low to very high stringency conditions,as described supra.

In a preferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 1. In another preferred aspect, thenucleic acid probe is nucleotides 388 to 1332 of SEQ ID NO: 1. Inanother preferred aspect, the nucleic acid probe is a polynucleotidesequence that encodes the polypeptide of SEQ ID NO: 2, or a subsequencethereof. In another preferred aspect, the nucleic acid probe is SEQ IDNO: 1. In another preferred aspect, the nucleic acid probe is thepolynucleotide sequence contained in plasmid pEJG120 which is containedin E. coli NRRL B-30699, wherein the polynucleotide sequence thereofencodes a polypeptide having cellulolytic enhancing activity. In anotherpreferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence contained in plasmid pEJG120 which is contained in E.coli NRRL B-30699.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 3. In another preferredaspect, the nucleic acid probe is nucleotides 98 to 821 of SEQ ID NO: 3.In another preferred aspect, the nucleic acid probe is a polynucleotidesequence that encodes the polypeptide of SEQ ID NO: 4, or a subsequencethereof. In another preferred aspect, the nucleic acid probe is SEQ IDNO: 3. In another preferred aspect, the nucleic acid probe is thepolynucleotide sequence contained in plasmid pTter61C which is containedin E. coli NRRL B-30813, wherein the polynucleotide sequence thereofencodes a polypeptide having cellulolytic enhancing activity. In anotherpreferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence contained in plasmid pTter61C which is contained in E.coli NRRL B-30813.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 5. In another preferredaspect, the nucleic acid probe is nucleotides 126 to 978 of SEQ ID NO:5. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 6, ora subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 5. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pTter61D whichis contained in E. coli NRRL B-30812, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pTter61D whichis contained in E. coli NRRL B-30812.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 7. In another preferredaspect, the nucleic acid probe is nucleotides 55 to 678 of SEQ ID NO: 7.In another preferred aspect, the nucleic acid probe is a polynucleotidesequence that encodes the polypeptide of SEQ ID NO: 8, or a subsequencethereof. In another preferred aspect, the nucleic acid probe is SEQ IDNO: 7. In another preferred aspect, the nucleic acid probe is thepolynucleotide sequence contained in plasmid pTter61E which is containedin E. coli NRRL B-30814, wherein the polynucleotide sequence thereofencodes a polypeptide having cellulolytic enhancing activity. In anotherpreferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence contained in plasmid pTter61E which is contained in E.coli NRRL B-30814.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 9. In another preferredaspect, the nucleic acid probe is nucleotides 58 to 912 of SEQ ID NO: 9In another preferred aspect, the nucleic acid probe is a polynucleotidesequence that encodes the polypeptide of SEQ ID NO: 10, or a subsequencethereof. In another preferred aspect, the nucleic acid probe is SEQ IDNO: 9. In another preferred aspect, the nucleic acid probe is thepolynucleotide sequence contained in plasmid pTter61G which is containedin E. coli NRRL B-30811, wherein the polynucleotide sequence thereofencodes a polypeptide having cellulolytic enhancing activity. In anotherpreferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence contained in plasmid pTter61G which is contained in E.coli NRRL B-30811.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 11. In another preferredaspect, the nucleic acid probe is nucleotides 46 to 951 of SEQ ID NO:11. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 12,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 11. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pTter61F whichis contained in E. coli NRRL B-50044, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding region contained in plasmid pTter61F which iscontained in E. coli NRRL B-50044.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 13. In another preferredaspect, the nucleic acid probe is nucleotides 67 to 796 of SEQ ID NO:13. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 14,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 13. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pDZA2-7 whichis contained in E. coli NRRL B-30704, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pDZA2-7 which iscontained in E. coli NRRL B-30704.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 15. In another preferredaspect, the nucleic acid probe is nucleotides 77 to 766 of SEQ ID NO:15. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 16,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 15. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pTr333 whichis contained in E. coli NRRL B-30878, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pTr333 which iscontained in E. coli NRRL B-30878.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures for 12 to 24 hours optimally.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at 45° C. (very low stringency), more preferably at50° C. (low stringency), more preferably at 55° C. (medium stringency),more preferably at 60° C. (medium-high stringency), even more preferablyat 65° C. (high stringency), and most preferably at 70° C. (very highstringency).

For short probes of about 15 nucleotides to about 70 nucleotides inlength, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(m), using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures for 12 to 24 hoursoptimally.

For short probes of about 15 nucleotides to about 70 nucleotides inlength, the carrier material is washed once in 6×SCC plus 0.1% SDS for15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C.below the calculated T_(m).

In a fifth aspect, the polypeptide having cellulolytic enhancingactivity is encoded by a polynucleotide comprising or consisting of anucleotide sequence that has a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO:15 of preferably at least 60%, more preferably at least 65%, morepreferably at least 70%, more preferably at least 75%, more preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, most preferably at least 95%, and even most preferably atleast 96%, at least 97%, at least 98%, or at least 99%.

In a preferred aspect, the mature polypeptide coding sequence isnucleotides 388 to 1332 of SEQ ID NO: 1, nucleotides 98 to 821 of SEQ IDNO: 3, nucleotides 126 to 978 of SEQ ID NO: 5, nucleotides 55 to 678 ofSEQ ID NO: 7, nucleotides 58 to 912 of SEQ ID NO: 9, nucleotides 46 to951 of SEQ ID NO: 11, nucleotides 67 to 796 of SEQ ID NO: 13, ornucleotides 77 to 766 of SEQ ID NO: 15.

In a sixth aspect, the polypeptide having cellulolytic enhancingactivity is an artificial variant comprising a substitution, deletion,and/or insertion of one or more (or several) amino acids of the maturepolypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, or SEQ ID NO: 16; or ahomologous sequence thereof. Methods for preparing such an artificialvariant is described supra.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14,or SEQ ID NO: 16, is 10, preferably 9, more preferably 8, morepreferably 7, more preferably at most 6, more preferably 5, morepreferably 4, even more preferably 3, most preferably 2, and even mostpreferably 1.

A polypeptide having cellulolytic enhancing activity may be obtainedfrom microorganisms of any genus. In a preferred aspect, the polypeptideobtained from a given source is secreted extracellularly.

A polypeptide having cellulolytic enhancing activity may be a bacterialpolypeptide. For example, the polypeptide may be a gram positivebacterial polypeptide such as a Bacillus, Streptococcus, Streptomyces,Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium,Geobacillus, or Oceanobacillus polypeptide having cellulolytic enhancingactivity, or a Gram negative bacterial polypeptide such as an E. coli,Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium,Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma polypeptide havingcellulolytic enhancing activity.

In a preferred aspect, the polypeptide is a Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having cellulolytic enhancing activity.

In another preferred aspect, the polypeptide is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus polypeptide having cellulolyticenhancing activity.

In another preferred aspect, the polypeptide is a Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans polypeptide havingcellulolytic enhancing activity.

The polypeptide having cellulolytic enhancing activity may also be afungal polypeptide, and more preferably a yeast polypeptide such as aCandida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia polypeptide having cellulolytic enhancing activity; or morepreferably a filamentous fungal polypeptide such as aan Acremonium,Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria,Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus,Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus,Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides,Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus,Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia,Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum,Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylariapolypeptide having cellulolytic enhancing activity.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide having cellulolyticenhancing activity.

In another preferred aspect, the polypeptide is an Acremoniumcellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillusfumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum,Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium zonatum, Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicolainsolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum,Penicillium purpurogenum, Phanerochaete chrysosporium, Thielaviaachromatica, Thielavia albomyces, Thielavia albopilosa, Thielaviaaustraleinsis, Thielavia fimeti, Thielavia microspora, Thielaviaovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa,Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,Trichoderma viride, or Trichophaea saccata polypeptide havingcellulolytic enhancing activity.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, polypeptides having cellulolytic enhancing activity may beidentified and obtained from other sources including microorganismsisolated from nature (e.g., soil, composts, water, etc.) using theabove-mentioned probes. Techniques for isolating microorganisms fromnatural habitats are well known in the art. The polynucleotide may thenbe obtained by similarly screening a genomic or cDNA library of such amicroorganism. Once a polynucleotide encoding a polypeptide has beendetected with the probe(s), the polynucleotide can be isolated or clonedby utilizing techniques that are well known to those of ordinary skillin the art (see, e.g., Sambrook et al., 1989, supra)

Polynucleotides comprising nucleotide sequences that encode polypeptidehaving cellulolytic enhancing activity can be isolated and utilized toexpress the polypeptide having cellulolytic enhancing activity forevaluation in the methods of the present invention, as described herein.

The polynucleotides comprise nucleotide sequences that have a degree ofidentity to the mature polypeptide coding sequence of SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQID NO: 13, or SEQ ID NO: 15 of preferably at least 60%, more preferablyat least 65%, more preferably at least 70%, more preferably at least75%, more preferably at least 80%, more preferably at least 85%, evenmore preferably at least 90%, most preferably at least 95%, and evenmost preferably at least 96%, at least 97%, at least 98%, or at least99%, which encode a polypeptide having cellulolytic enhancing activity.

The polynucleotide may also be a polynucleotide encoding a polypeptidehaving cellulolytic enhancing activity that hybridizes under at leastvery low stringency conditions, preferably at least low stringencyconditions, more preferably at least medium stringency conditions, morepreferably at least medium-high stringency conditions, even morepreferably at least high stringency conditions, and most preferably atleast very high stringency conditions with (i) t the mature polypeptidecoding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15, (ii)the cDNA sequence contained in the mature polypeptide coding sequence ofSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 13, or thegenomic DNA sequence comprising the mature polypeptide coding sequenceof SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 15, or (iii)a full-length complementary strand of (i) or (ii); or allelic variantsand subsequences thereof (Sambrook et al., 1989, supra), as definedherein. In a preferred aspect, the mature polypeptide coding sequence isnucleotides 388 to 1332 of SEQ ID NO: 1, nucleotides 98 to 821 of SEQ IDNO: 3, nucleotides 126 to 978 of SEQ ID NO: 5, nucleotides 55 to 678 ofSEQ ID NO: 7, nucleotides 58 to 912 of SEQ ID NO: 9, nucleotides 46 to951 of SEQ ID NO: 11, nucleotides 67 to 796 of SEQ ID NO: 13, ornucleotides 77 to 766 of SEQ ID NO: 15.

As described earlier, the techniques used to isolate or clone apolynucleotide encoding a polypeptide are known in the art and includeisolation from genomic DNA, preparation from cDNA, or a combinationthereof.

Cellobiose Dehydrogenases and Polynucleotides Thereof

In the present invention, a cellobiose dehydrogenase may be present asan enzyme activity in the enzyme composition and/or as a component ofone or more protein components added to the composition. The cellobiosedehydrogenase can be any cellobiose dehydrogenase.

The cellobiose dehydrogenase may be obtained from microorganisms of anygenus. For purposes of the present invention, the term “obtained from”as used herein in connection with a given source shall mean that thepolypeptide encoded by a nucleotide sequence is produced by the sourceor by a strain in which the nucleotide sequence from the source has beeninserted. In one aspect, the polypeptide obtained from a given source issecreted extracellularly.

The cellobiose dehydrogenase may be a bacterial polypeptide. Forexample, the polypeptide may be a gram positive bacterial polypeptidesuch as a Bacillus, Streptococcus, Streptomyces, Staphylococcus,Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, orOceanobacillus cellobiose dehydrogenase, or a Gram negative bacterialpolypeptide such as an E. coli, Pseudomonas, Salmonella, Campylobacter,Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, orUreaplasma cellobiose dehydrogenase.

In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis cellobiose dehydrogenase.

In another aspect, the polypeptide is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus cellobiose dehydrogenase.

In another aspect, the polypeptide is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans cellobiose dehydrogenase.

The cellobiose dehydrogenase may also be a fungal polypeptide, and morepreferably a yeast polypeptide such as a Candida, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cellobiosedehydrogenase; or more preferably a filamentous fungal polypeptide suchas an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium,Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps,Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria,Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella,Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria,Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete,Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor,Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia,Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, orXylaria cellobiose dehydrogenase.

In another aspect, the polypeptide is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis cellobiose dehydrogenase.

In another aspect, the polypeptide is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus,Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum,Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporiummerdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielaviaspededonium, Thielavia setosa, Thielavia subthermophila, Thielaviaterrestris, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride cellobiosedehydrogenase.

Examples of other cellobiose dehydrogenases and their sources are listedin Table 1.

TABLE 1 Published microbial cellobiose dehydrogenase sequences AccessionSpecies # Literature Reference Humicola insolens Q9P8H5 Xu et al., 2001,Humicola insolens cellobiose dehydrogenase: cloning, redox chemistry,and “logic gate”-like dual functionality, Enz. Microb. Technol. 28:744-753 Irpex lacteus Q6AW20 Nozaki et al., 1999, Cloning and expressionof cellobiose dehydrogenase from Irpex lacteus. Submitted (AUG-2004) tothe EMBL/GenBank/DDBJ databases. Pycnoporus O74253 Moukha et al., 1999,Cloning and analysis cinnabarinus of Pycnoporus cinnabarinus cellobiosedehydrogenase, Gene 234: 23-33 Phanerochaete Q01738 Li et al., 1996,Cloning of a cDNA chrysosporium encoding cellobiose dehydrogenase, ahemoflavoenzyme from Phanerochaete chrysosporium, Appl. Environ.Microbiol. 62: 1329-1335 Coniophora Q6BDD5 Kajisa et al., 2004,Characterization and puteana molecular cloning of cellobiosedehydrogenase from the brown-rot fungus Coniophora puteana, Biosci.Bioeng. 98: 57-63 Athelia rolfsii Q7Z975 Zamocky et al., Phylogeneticanalysis of cellobiose dehydrogenases. Submitted (NOV-2002) to theEMBL/GenBank/DDBJ databases Grifola Q8J2T4 Yoshida et al., 2002,Molecular cloning frondosa and characterization of a cDNA encodingcellobiose dehydrogenase from the wood- rotting fungus Grifola frondosa,FEMS Microbiol. Lett. 217: 225-230 Trametes Q875J3 Stapleton et al.,2004, Molecular cloning of versicolor the cellobiose dehydrogenase genefrom Trametes versicolor and expression in Pichia pastoris, EnzymeMicrob. Technol. 34: 55-63 Trametes O42729 Dumonceaux et al., 1998,Cloning and versicolor sequencing of a gene encoding cellobiosedehydrogenase from Trametes versicolor, Gene 210: 211-219 AspergillusQ4WIN9 Nierman et al., 2005, Genomic sequence of fumigatus thepathogenic and allergenic filamentous fungus Aspergillus fumigatus,Nature 438: 1151-1156 Phanerochaete Q12661 Raices et al., 1995, Cloningand chrysosporium characterization of a cDNA encoding a cellobiosedehydrogenase from the white rot fungus Phanerochaete chrysosporium,FEBS Lett. 369: 233-238 Myriococcum A9XK88 Zamocky et al., 2008,Cloning, sequence thermophilum analysis and heterologous expression inPichia pastoris of a gene encoding a thermostable cellobiosedehydrogenase from Myriococcum thermophilum, Protein Expr. Purif. 59:258-265 Aspergillus A1CFV0 Fedorova et al., Genomic islands in theclavatus pathogenic filamentous fungus Aspergillus fumigatus, PLoSCorynascus O74240 Subramaniam et al., Biochemical and heterothallicusmolecular biological characterization of cellobiose dehydrogenase fromSporotrichum thermophile. Submitted (JUN-1998) to the EMBL/GenBank/DDBJdatabases Neosartorya A1CYG2 Fedorova et al., Genomic islands in thefischeri pathogenic filamentous fungus Aspergillus fumigatus, PLoSAspergillus B0XVQ8 Fedorova et al., Genomic islands in the fumigatuspathogenic filamentous fungus Aspergillus fumigatus, PLoS AspergillusA1C890 Fedorova et al., Genomic islands in the clavatus pathogenicfilamentous fungus Aspergillus fumigatus, PLoS Neosartorya A1DIY3Fedorova et al., Genomic islands in the fischeri pathogenic filamentousfungus Aspergillus fumigatus, PLoS Myriococcum A9XK87 Zamocky et al.,2008, Cloning, sequence thermophilum analysis and heterologousexpression in Pichia pastoris of a gene encoding a thermostablecellobiose dehydrogenase from Myriococcum thermophilum, Protein Expr.Purif. 59: 258-265 Pyrenophora B2WHI7 Birren et al., The Broad InstituteGenome tritici-repentis Sequencing Plafform “Genome Sequence ofPyrenophora tritici-repentis. Submitted (MAR-2007) to theEMBL/GenBank/DDBJ databases Pyrenophora B2WJX3 Birren et al., The BroadInstitute Genome tritici-repentis Sequencing Platform “Genome Sequenceof Pyrenophora tritici-repentis. Submitted (MAR-2007) to theEMBL/GenBank/DDBJ databases Aspergillus Q4WC40 Fedorova et al., Genomicislands in the fumigatus pathogenic filamentous fungus Aspergillusfumigatus, PLoS Aspergillus A2QD75 Pel et al., 2007, Genome sequencingniger and analysis of the versatile cell factory Aspergillus niger CBS513.88, Nat. Biotechnol. 25: 221-231

In another aspect, the cellobiose dehydrogenase is a Humicola insolenscellobiose dehydrogenase. In another aspect, the cellobiosedehydrogenase is a Humicola insolens DSM 1800 cellobiose dehydrogenase,e.g., the polypeptide comprising SEQ ID NO: 18 encoded by SEQ ID NO: 17,or a fragment thereof having cellobiose dehydrogenase activity (see U.S.Pat. No. 6,280,976).

In another aspect, the cellobiose dehydrogenase is a Myceliophthorathermophila cellobiose dehydrogenase. In another aspect, the cellobiosedehydrogenase is a Myceliophthora thermophila CBS 117.65 cellobiosedehydrogenase.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

As described earlier, the techniques used to isolate or clone apolynucleotide encoding a polypeptide are known in the art and includeisolation from genomic DNA, preparation from cDNA, or a combinationthereof.

Enzyme Compositions

In the methods of the present invention, the enzyme composition maycomprise any protein involved in the processing of acellulose-containing material to glucose and/or cellobiose, orhemicellulose to xylose, mannose, galactose, and/or arabinose.

The enzyme composition preferably comprises enzymes having cellulolyticactivity and/or xylan degrading activity. In one aspect, the enzymecomposition comprises one or more (several) cellulolytic enzymes. Inanother aspect, the enzyme composition comprises one or more (several)xylan degrading enzymes. In another aspect, the enzyme compositioncomprises one or more (several) cellulolytic enzymes and one or more(several) xylan degrading enzymes.

The one or more (several) cellulolytic enzymes are preferably selectedfrom the group consisting of an endoglucanase, a cellobiohydrolase, anda beta-glucosidase. The one or more (several) xylan degrading enzymesare preferably selected from the group consisting of a xylanase, anacetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

In another aspect, the enzyme composition further or even furthercomprises a polypeptide having cellulolytic enhancing activity (see, forexample, WO 2005/074647, WO 2005/074656, and WO 2007/089290). In anotheraspect, the enzyme composition may further or even further comprise oneor more (several) additional enzyme activities to improve thedegradation of the cellulose-containing material. Preferred additionalenzymes are hemicellulases (e.g., alpha-D-glucuronidases,alpha-L-arabinofuranosidases, endo-mannanases, beta-mannosidases,alpha-galactosidases, endo-alpha-L-arabinanases, beta-galactosidases),carbohydrate-esterases (e.g., acetyl-xylan esterases, acetyl-mannanesterases, ferulic acid esterases, coumaric acid esterases, glucuronoylesterases), pectinases, proteases, ligninolytic enzymes (e.g., laccases,manganese peroxidases, lignin peroxidases, H₂O₂-producing enzymes,oxidoreductases), expansins, swollenins, or mixtures thereof. In themethods of the present invention, the additional enzyme(s) can be addedprior to or during fermentation, e.g., during saccharification or duringor after propagation of the fermenting microorganism(s).

One or more (several) components of the enzyme composition may bewild-type proteins, recombinant proteins, or a combination of wild-typeproteins and recombinant proteins. For example, one or more (several)components may be native proteins of a cell, which is used as a hostcell to express recombinantly one or more (several) other components ofthe enzyme composition. One or more (several) components of the enzymecomposition may be produced as monocomponents, which are then combinedto form the enzyme composition. The enzyme composition may be acombination of multicomponent and monocomponent protein preparations.

The enzymes used in the methods of the present invention may be in anyform suitable for use in the processes described herein, such as, forexample, a crude fermentation broth with or without cells removed, acell lysate with or without cellular debris, a semi-purified or purifiedenzyme preparation, or a host cell as a source of the enzymes. Theenzyme composition may be a dry powder or granulate, a non-dustinggranulate, a liquid, a stabilized liquid, or a stabilized protectedenzyme. Liquid enzyme preparations may, for instance, be stabilized byadding stabilizers such as a sugar, a sugar alcohol or another polyol,and/or lactic acid or another organic acid according to establishedprocesses.

A polypeptide having cellulolytic enzyme activity or xylan degradingactivity may be a bacterial polypeptide. For example, the polypeptidemay be a gram positive bacterial polypeptide such as a Bacillus,Streptococcus, Streptomyces, Staphylococcus, Enterococcus,Lactobacillus, Lactococcus, Clostridium, Geobacillus, or Oceanobacilluspolypeptide having cellulolytic enzyme activity or xylan degradingactivity, or a Gram negative bacterial polypeptide such as an E. coli,Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium,Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma polypeptide havingcellulolytic enzyme activity or xylan degrading activity.

In a preferred aspect, the polypeptide is a Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having cellulolytic enzyme activity or xylandegrading activity.

In another preferred aspect, the polypeptide is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus polypeptide having cellulolyticenzyme activity or xylan degrading activity.

In another preferred aspect, the polypeptide is a Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans polypeptide havingcellulolytic enzyme activity or xylan degrading activity.

The polypeptide having cellulolytic enzyme activity or xylan degradingactivity may also be a fungal polypeptide, and more preferably a yeastpolypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia polypeptide having cellulolytic enzymeactivity or xylan degrading activity; or more preferably a filamentousfungal polypeptide such as an Acremonium, Agaricus, Alternaria,Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide having cellulolyticenzyme activity or xylan degrading activity.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide having cellulolyticenzyme activity or xylan degrading activity.

In another preferred aspect, the polypeptide is an Acremoniumcellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillusfumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum,Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium zonatum, Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicolainsolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum,Penicillium purpurogenum, Phanerochaete chrysosporium, Thielaviaachromatica, Thielavia albomyces, Thielavia albopilosa, Thielaviaaustraleinsis, Thielavia fimeti, Thielavia microspora, Thielaviaovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa,Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,Trichoderma viride, or Trichophaea saccata polypeptide havingcellulolytic enzyme activity or xylan degrading activity.

Chemically modified or protein engineered mutants of polypeptides havingcellulolytic enzyme activity or xylan degrading activity may also beused.

One or more (several) components of the enzyme composition may be arecombinant component, i.e., produced by cloning of a DNA sequenceencoding the single component and subsequent cell transformed with theDNA sequence and expressed in a host (see, for example, WO 91/17243 andWO 91/17244). The host is preferably a heterologous host (enzyme isforeign to host), but the host may under certain conditions also be ahomologous host (enzyme is native to host). Monocomponent cellulolyticproteins may also be prepared by purifying such a protein from afermentation broth.

Examples of commercial cellulolytic protein preparations suitable foruse in the present invention include, for example, CELLIC™ Ctec(Novozymes A/S), CELLUCLAST™ (Novozymes A/S), NOVOZYM™ 188 (NovozymesA/S), CELLUZYME™ (Novozymes A/S), CEREFLO™ (Novozymes A/S), andULTRAFLO™ (Novozymes A/S), ACCELERASE™ (Genencor Int.), LAMINEX™(Genencor Int.), SPEZYME™ CP (Genencor Int.), ROHAMENT™ 7069 W (RöhmGmbH), FIBREZYME® LDI (Dyadic International, Inc.), FIBREZYME® LBR(Dyadic International, Inc.), or VISCOSTAR® 150 L (Dyadic International,Inc.). The cellulase enzymes are added in amounts effective from about0.001 to about 5.0 wt % of solids, more preferably from about 0.025 toabout 4.0 wt % of solids, and most preferably from about 0.005 to about2.0 wt % of solids. The cellulase enzymes are added in amounts effectivefrom about 0.001 to about 5.0 wt % of solids, more preferably from about0.025 to about 4.0 wt % of solids, and most preferably from about 0.005to about 2.0 wt % of solids.

Examples of bacterial endoglucanases that can be used in the methods ofthe present invention, include, but are not limited to, an Acidothermuscellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No.5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO 00/70031, WO05/093050); Thermobifida fusca endoglucanase III (WO 05/093050); andThermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the methods of thepresent invention, include, but are not limited to, a Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263; GENBANK™accession no. M15665); Trichoderma reesei endoglucanase II (Saloheimo,et al., 1988, Gene 63:11-22; GENBANK™ accession no. M19373); Trichodermareesei endoglucanase III (Okada et al., 1988, Appl. Environ. Microbiol.64: 555-563; GENBANK™ accession no. AB003694); Trichoderma reeseiendoglucanase IV (Saloheimo et al., 1997, Eur. J. Biochem. 249: 584-591;GENBANK™ accession no. Y11113); and Trichoderma reesei endoglucanase V(Saloheimo et al., 1994, Molecular Microbiology 13: 219-228; GENBANK™accession no. Z33381); Aspergillus aculeatus endoglucanase (Ooi et al.,1990, Nucleic Acids Research 18: 5884); Aspergillus kawachiiendoglucanase (Sakamoto et al., 1995, Current Genetics 27: 435-439);Erwinia carotovara endoglucanase (Saarilahti et al., 1990, Gene 90:9-14); Fusarium oxysporum endoglucanase (GENBANK™ accession no. L29381);Humicola grisea var. thermoidea endoglucanase (GENBANK™ accession no.AB003107); Melanocarpus albomyces endoglucanase (GENBANK™ accession no.MAL515703); Neurospora crassa endoglucanase (GENBANK™ accession no.XM_(—)324477); Humicola insolens endoglucanase V (SEQ ID NO: 20);Myceliophthora thermophila CBS 117.65 endoglucanase (SEQ ID NO: 22);basidiomycete CBS 495.95 endoglucanase (SEQ ID NO: 24); basidiomyceteCBS 494.95 endoglucanase (SEQ ID NO: 26); Thielavia terrestris NRRL 8126CEL6B endoglucanase (SEQ ID NO: 28); Thielavia terrestris NRRL 8126CEL6C endoglucanase (SEQ ID NO: 30); Thielavia terrestris NRRL 8126CEL7C endoglucanase (SEQ ID NO: 32); Thielavia terrestris NRRL 8126CEL7E endoglucanase (SEQ ID NO: 34); Thielavia terrestris NRRL 8126CEL7F endoglucanase (SEQ ID NO: 36); Cladorrhinum foecundissimum ATCC62373 CEL7A endoglucanase (SEQ ID NO: 38); and Trichoderma reesei strainNo. VTT-D-80133 endoglucanase (SEQ ID NO: 40; GENBANK™ accession no.M15665). The endoglucanases of SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ IDNO: 34, SEQ ID NO: 36, SEQ ID NO: 38, and SEQ ID NO: 40 described aboveare encoded by the mature polypeptide coding sequence of SEQ ID NO: 19,SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO:29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ IDNO: 39, respectively.

Examples of cellobiohydrolases useful in the methods of the presentinvention include, but are not limited to, Trichoderma reeseicellobiohydrolase I (SEQ ID NO: 42); Trichoderma reeseicellobiohydrolase II (SEQ ID NO: 44); Humicola insolenscellobiohydrolase I (SEQ ID NO: 46), Myceliophthora thermophilacellobiohydrolase II (SEQ ID NO: 48 and SEQ ID NO: 50), Thielaviaterrestris cellobiohydrolase II (CEL6A) (SEQ ID NO: 52), Chaetomiumthermophilum cellobiohydrolase I (SEQ ID NO: 54), and Chaetomiumthermophilum cellobiohydrolase II (SEQ ID NO: 56). Thecellobiohydrolases of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, and SEQ ID NO:54 described above are encoded by the mature polypeptide coding sequenceof SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ IDNO: 49, SEQ ID NO: 51, SEQ ID NO: 53, and SEQ ID NO: 55, respectively.

Examples of beta-glucosidases useful in the methods of the presentinvention include, but are not limited to, Aspergillus oryzaebeta-glucosidase (SEQ ID NO: 58); Aspergillus fumigatus beta-glucosidase(SEQ ID NO: 60); Penicillium brasilianum IBT 20888 beta-glucosidase (SEQID NO: 62); Aspergillus niger beta-glucosidase (SEQ ID NO: 64); andAspergillus aculeatus beta-glucosidase (SEQ ID NO: 66). Thebeta-glucosidases of SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ IDNO: 64, and SEQ ID NO: 66 described above are encoded by the maturepolypeptide coding sequence of SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO:61, SEQ ID NO: 63, and SEQ ID NO: 65, respectively.

The Aspergillus oryzae polypeptide having beta-glucosidase activity canbe obtained according to WO 2002/095014. The Aspergillus fumigatuspolypeptide having beta-glucosidase activity can be obtained accordingto WO 2005/047499. The Penicillium brasilianum polypeptide havingbeta-glucosidase activity can be obtained according to WO 2007/019442.The Aspergillus niger polypeptide having beta-glucosidase activity canbe obtained according to Dan et al., 2000, J. Biol. Chem. 275:4973-4980. The Aspergillus aculeatus polypeptide having beta-glucosidaseactivity can be obtained according to Kawaguchi et al., 1996, Gene 173:287-288.

The beta-glucosidase may be a fusion protein. In one aspect, thebeta-glucosidase is the Aspergillus oryzae beta-glucosidase variant BGfusion protein of SEQ ID NO: 68 or the Aspergillus oryzaebeta-glucosidase fusion protein of SEQ ID NO: 70. In another aspect, theAspergillus oryzae beta-glucosidase variant BG fusion protein is encodedby the polynucleotide of SEQ ID NO: 67 or the Aspergillus oryzaebeta-glucosidase fusion protein is encoded by the polynucleotide of SEQID NO: 69.

Other endoglucanases, cellobiohydrolases, and beta-glucosidases aredisclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat B., 1991, A classification ofglycosyl hydrolases based on amino-acid sequence similarities, Biochem.J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating thesequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696.

Other cellulolytic enzymes that may be used in the present invention aredescribed in EP 495,257, EP 531,315, EP 531,372, WO 89/09259, WO94/07998, WO 95/24471, WO 96/11262, WO 96/29397, WO 96/034108, WO97/14804, WO 98/08940, WO 98/012307, WO 98/13465, WO 98/015619, WO98/015633, WO 98/028411, WO 99/06574, WO 99/10481, WO 99/025846, WO99/025847, WO 99/031255, WO 2000/009707, WO 2002/050245, WO2002/0076792, WO 2002/101078, WO 2003/027306, WO 2003/052054, WO2003/052055, WO 2003/052056, WO 2003/052057, WO 2003/052118, WO2004/016760, WO 2004/043980, WO 2004/048592, WO 2005/001065, WO2005/028636, WO 2005/093050, WO 2005/093073, WO 2006/074005, WO2006/117432, WO 2007/071818, WO 2007/071820, WO 2008/008070, WO2008/008793, U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,457,046, U.S. Pat.No. 5,648,263, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,691,178, U.S.Pat. No. 5,763,254, and U.S. Pat. No. 5,776,757.

Examples of commercial xylan degrading enzyme preparations suitable foruse in the present invention include, for example, SHEARZYME™ (NovozymesA/S), CELLIC™ Htec (Novozymes A/S), VISCOZYME® (Novozymes A/S),ULTRAFLO® (Novozymes A/S), PULPZYME® HC (Novozymes A/S), MULTIFECT®Xylanase (Genencor), ECOPULP® TX-200A (AB Enzymes), HSP 6000 Xylanase(DSM), DEPOL™ 333P (Biocatalysts Limit, Wales, UK), DEPOL™ 740 L.(Biocatalysts Limit, Wales, UK), and DEPOL™ 762P (Biocatalysts Limit,Wales, UK).

Examples of xylanases useful in the methods of the present inventioninclude, but are not limited to, Aspergillus aculeatus xylanase(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus xylanases (WO2006/078256), and Thielavia terrestris NRRL 8126 xylanases (WO2009/079210).

Examples of beta-xylosidases useful in the methods of the presentinvention include, but are not limited to, Trichoderma reeseibeta-xylosidase (UniProtKB/TrEMBL accession number Q92458), Talaromycesemersonii (SwissProt accession number Q8×212), and Neurospora crassa(SwissProt accession number Q7SOW4).

Examples of acetylxylan esterases useful in the methods of the presentinvention include, but are not limited to, Hypocrea jecorina acetylxylanesterase (WO 2005/001036), Neurospora crassa acetylxylan esterase(UniProt accession number q7s259), Thielavia terrestris NRRL 8126acetylxylan esterase (WO 2009/042846), Chaetomium globosum acetylxylanesterase (Uniprot accession number Q2GWX4), Chaetomium gracileacetylxylan esterase (GeneSeqP accession number AAB82124), Phaeosphaerianodorum acetylxylan esterase (Uniprot accession number Q0UHJ1), andHumicola insolens DSM 1800 acetylxylan esterase (WO 2009/073709).

Examples of ferulic acid esterases useful in the methods of the presentinvention include, but are not limited to, Humicola insolens DSM 1800feruloyl esterase (WO 2009/076122), Neurospora crassa feruloyl esterase(UniProt accession number Q9HGR3), and Neosartorya fischeri feruloylesterase (UniProt Accession number A1D9T4).

Examples of arabinofuranosidases useful in the methods of the presentinvention include, but are not limited to, Humicola insolens DSM 1800arabinofuranosidase (WO 2009/073383) and Aspergillus nigerarabinofuranosidase (GeneSeqP accession number AAR94170).

Examples of alpha-glucuronidases useful in the methods of the presentinvention include, but are not limited to, Aspergillus clavatusalpha-glucuronidase (UniProt accession number alcc12), Trichodermareesei alpha-glucuronidase (Uniprot accession number Q99024),Talaromyces emersonii alpha-glucuronidase (UniProt accession numberQ8×211), Aspergillus niger alpha-glucuronidase (Uniprot accession numberQ96WX9), Aspergillus terreus alpha-glucuronidase (SwissProt accessionnumber Q0CJP9), and Aspergillus fumigatus alpha-glucuronidase (SwissProtaccession number Q4WW45).

The enzymes and proteins used in the methods of the present inventionmay be produced by fermentation of the above-noted microbial strains ona nutrient medium containing suitable carbon and nitrogen sources andinorganic salts, using procedures known in the art (see, e.g., Bennett,J. W. and LaSure, L. (eds.), More Gene Manipulations in Fungi, AcademicPress, CA, 1991). Suitable media are available from commercial suppliersor may be prepared according to published compositions (e.g., incatalogues of the American Type Culture Collection). Temperature rangesand other conditions suitable for growth and enzyme production are knownin the art (see, e.g., Bailey, J. E., and 011 is, D. F., BiochemicalEngineering Fundamentals, McGraw-Hill Book Company, NY, 1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of an enzyme. Fermentation may, therefore,be understood as comprising shake flask cultivation, or small- orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the enzymeto be expressed or isolated. The resulting enzymes produced by themethods described above may be recovered from the fermentation mediumand purified by conventional procedures.

Nucleic Acid Constructs

Nucleic acid constructs comprising an isolated polynucleotide encoding apolypeptide of interest, e.g., polypeptide having cellulolytic enhancingactivity or one or more (several) components of the enzyme composition,operably linked to one or more (several) control sequences may beconstructed that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences.

The isolated polynucleotide may be manipulated in a variety of ways toprovide for expression of the polypeptide. Manipulation of thepolynucleotide's sequence prior to its insertion into a vector may bedesirable or necessary depending on the expression vector. Thetechniques for modifying polynucleotide sequences utilizing recombinantDNA methods are well known in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence that is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide of the present invention. Thepromoter sequence contains transcriptional control sequences thatmediate the expression of the polypeptide. The promoter may be anynucleotide sequence that shows transcriptional activity in the host cellof choice including mutant, truncated, and hybrid promoters, and may beobtained from genes encoding extracellular or intracellular polypeptideseither homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs in a filamentous fungal host cell are promotersobtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucormiehei aspartic proteinase, Aspergillus niger neutral alpha-amylase,Aspergillus niger acid stable alpha-amylase, Aspergillus niger orAspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase,Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dana (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporumtrypsin-like protease (WO 96/00787), Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter including a gene encoding a neutralalpha-amylase in Aspergilli in which the untranslated leader has beenreplaced by an untranslated leader from a gene encoding triose phosphateisomerase in Aspergilli; non-limiting examples include modifiedpromoters including the gene encoding neutral alpha-amylase inAspergillus niger in which the untranslated leader has been replaced byan untranslated leader from the gene encoding triose phosphate isomerasein Aspergillus nidulans or Aspergillus oryzae); and mutant, truncated,and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator that is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding sequence thatencodes a signal peptide linked to the amino terminus of a polypeptideand directs the encoded polypeptide into the cell's secretory pathway.The 5′ end of the coding sequence of the nucleotide sequence mayinherently contain a signal peptide coding sequence naturally linked intranslation reading frame with the segment of the coding sequence thatencodes the secreted polypeptide. Alternatively, the 5′ end of thecoding sequence may contain a signal peptide coding sequence that isforeign to the coding sequence. The foreign signal peptide codingsequence may be required where the coding sequence does not naturallycontain a signal peptide coding sequence. Alternatively, the foreignsignal peptide coding sequence may simply replace the natural signalpeptide coding sequence in order to enhance secretion of thepolypeptide. However, any signal peptide coding sequence that directsthe expressed polypeptide into the secretory pathway of a host cell ofchoice, i.e., secreted into a culture medium, may be used in the presentinvention.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus stearothermophilusalpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, Humicola insolens endoglucanase V, andHumicola lanuginosa lipase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the amino terminus of a polypeptide.The resultant polypeptide is known as a proenzyme or propolypeptide (ora zymogen in some cases). A propeptide is generally inactive and can beconverted to a mature active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei asparticproteinase, and Myceliophthora thermophila laccase (WO 95/33836).

Where both signal peptide and propeptide sequences are present at theamino terminus of a polypeptide, the propeptide sequence is positionednext to the amino terminus of a polypeptide and the signal peptidesequence is positioned next to the amino terminus of the propeptidesequence.

It may also be desirable to add regulatory sequences that allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those that causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the nucleotide sequence encoding thepolypeptide would be operably linked with the regulatory sequence.

Expression Vectors

Recombinant expression vectors comprising a polynucleotide encoding apolypeptide of interest, a promoter, and transcriptional andtranslational stop signals may be constructed for expression of thepolypeptide in a suitable host cell. The various nucleic acids andcontrol sequences described herein may be joined together to produce arecombinant expression vector that may include one or more (several)convenient restriction sites to allow for insertion or substitution ofthe nucleotide sequence encoding the polypeptide at such sites.Alternatively, a polynucleotide sequence may be expressed by insertingthe nucleotide sequence or a nucleic acid construct comprising thesequence into an appropriate vector for expression. In creating theexpression vector, the coding sequence is located in the vector so thatthe coding sequence is operably linked with the appropriate controlsequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the nucleotide sequence. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vectors preferably contain one or more (several) selectable markersthat permit easy selection of transformed, transfected, transduced, orthe like cells. A selectable marker is a gene the product of whichprovides for biocide or viral resistance, resistance to heavy metals,prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers that confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors preferably contain an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of identity to the correspondingtarget sequence to enhance the probability of homologous recombination.The integrational elements may be any sequence that is homologous withthe target sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleotidesequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide may be inserted into a host cellto increase production of the gene product. An increase in the copynumber of the polynucleotide can be obtained by integrating at least oneadditional copy of the sequence into the host cell genome or byincluding an amplifiable selectable marker gene with the polynucleotidewhere cells containing amplified copies of the selectable marker gene,and thereby additional copies of the polynucleotide, can be selected forby cultivating the cells in the presence of the appropriate selectableagent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors are well known to one skilled in theart (see, e.g., Sambrook et al., 1989, supra).

Host Cells

The nucleic acid constructs or expression vectors comprising an isolatedpolynucleotide encoding a polypeptide of interest may be introduced intorecombinant host cells for the recombinant production of thepolypeptides. A vector comprising a polynucleotide is introduced into ahost cell so that the vector is maintained as a chromosomal integrant oras a self-replicating extra-chromosomal vector as described earlier. Theterm “host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram positive bacterium or a Gramnegative bacterium. Gram positive bacteria include, but not limited to,Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus,Lactobacillus, Lactococcus, Clostridium, Geobacillus, andOceanobacillus. Gram negative bacteria include, but not limited to, E.coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter,Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, and Ureaplasma.

The bacterial host cell may be any Bacillus cell. Bacillus cellsinclude, but are not limited to, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, and Bacillusthuringiensis cells.

In a preferred aspect, the bacterial host cell is a Bacillusamyloliquefaciens, Bacillus lentus, Bacillus licheniformis, Bacillusstearothermophilus or Bacillus subtilis cell. In a more preferredaspect, the bacterial host cell is a Bacillus amyloliquefaciens cell. Inanother more preferred aspect, the bacterial host cell is a Bacillusclausii cell. In another more preferred aspect, the bacterial host cellis a Bacillus licheniformis cell. In another more preferred aspect, thebacterial host cell is a Bacillus subtilis cell.

The bacterial host cell may also be any Streptococcus cell.Streptococcus cells include, but are not limited to, Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, andStreptococcus equi subsp. Zooepidemicus cells.

In a preferred aspect, the bacterial host cell is a Streptococcusequisimilis cell. In another preferred aspect, the bacterial host cellis a Streptococcus pyogenes cell. In another preferred aspect, thebacterial host cell is a Streptococcus uberis cell. In another preferredaspect, the bacterial host cell is a Streptococcus equi subsp.Zooepidemicus cell.

The bacterial host cell may also be any Streptomyces cell. Streptomycescells include, but are not limited to, Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,and Streptomyces lividans cells.

In a preferred aspect, the bacterial host cell is a Streptomycesachromogenes cell. In another preferred aspect, the bacterial host cellis a Streptomyces avermitilis cell. In another preferred aspect, thebacterial host cell is a Streptomyces coelicolor cell. In anotherpreferred aspect, the bacterial host cell is a Streptomyces griseuscell. In another preferred aspect, the bacterial host cell is aStreptomyces lividans cell.

The introduction of DNA into a Bacillus cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Molecular General Genetics 168: 111-115), by using competent cells (see,e.g., Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829, orDubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988,Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5271-5278). The introductionof DNA into an E. coli cell may, for instance, be effected by protoplasttransformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) orelectroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). The introduction of DNA into a Streptomyces cell may, forinstance, be effected by protoplast transformation and electroporation(see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), byconjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or by transduction (see, e.g., Burke et al., 2001, Proc.Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may, for instance, be effected by electroporation (see,e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or byconjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ.Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cellmay, for instance, be effected by natural competence (see, e.g., Perryand Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), by protoplasttransformation (see, e.g., Catt and Jollick, 1991, Microbios. 68:189-207, by electroporation (see, e.g., Buckley et al., 1999, Appl.Environ. Microbiol. 65: 3800-3804) or by conjugation (see, e.g.,Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method knownin the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota (as defined by Hawksworth et al., In, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred aspect, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis cell. In another most preferredaspect, the yeast host cell is a Kluyveromyces lactis cell. In anothermost preferred aspect, the yeast host cell is a Yarrowia lipolyticacell.

In another more preferred aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred aspect, the filamentous fungal host cell is anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium,Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia,Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus,Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is anAspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger orAspergillus oryzae cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another most preferred aspect, the filamentous fungalhost cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsisaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens,Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,Ceriporiopsis subvermispora, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium tropicum, Chrysosporium merdarium,Chrysosporium inops, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Coprinus cinereus, Coriolushirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

A polypeptide of interest, e.g., polypeptide having cellulolyticenhancing activity or one or more (several) cellulolytic enzymes, can beproduced by (a) cultivating a cell, which in its wild-type form producesthe polypeptide, under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide.

The polypeptide of interest can also be produced by (a) cultivating arecombinant host cell, as described herein, under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.

In the production methods, the cells are cultivated in a nutrient mediumsuitable for production of the polypeptide using methods well known inthe art. For example, the cell may be cultivated by shake flaskcultivation, and small-scale or large-scale fermentation (includingcontinuous, batch, fed-batch, or solid state fermentations) inlaboratory or industrial fermentors performed in a suitable medium andunder conditions allowing the polypeptide to be expressed and/orisolated. The cultivation takes place in a suitable nutrient mediumcomprising carbon and nitrogen sources and inorganic salts, usingprocedures known in the art. Suitable media are available fromcommercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted into the medium, it can be recovered fromcell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide as described herein.

The resulting polypeptide may be recovered using methods known in theart. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989) to obtainsubstantially pure polypeptides.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Example 1 Growth of Myceliophthora thermophila CBS 117.65

Two plugs from a PDA plate of Myceliophthora thermophila CBS 117.65 wereinoculated into a 500 ml shake flask containing 100 ml of shake flaskmedium to obtain culture broth for the purification of a cellobiosedehydrogenase. PDA plates were composed of 39 g of potato dextrose agarand deionized water to 1 liter. The shake flask medium was composed of15 g of glucose, 4 g of K₂HPO₄, 1 g of NaCl, 0.2 g of MgSO₄.7H₂O, 2 g ofMES free acid, 1 g of Bacto Peptone, 5 g of yeast extract, 2.5 g ofcitric acid, 0.2 g of CaCl₂.2H₂O, 5 g of NH₄NO₃, 1 ml of trace elementssolution, and deionized water to 1 liter. The trace elements solutionwas composed of 1.2 g of FeSO₄.7H₂O, 10 g of ZnSO₄.7H₂O, 0.7 g ofMnSO₄.H₂O, 0.4 g of CuSO₄.5H₂O, 0.4 g of Na₂B₄O₇.10H₂O, 0.8 g ofNa₂MoO₂.2H₂O, and deionized water to 1 liter. The shake flask wasincubated at 45° C. on an orbital shaker at 200 rpm for 48 hours. Fiftyml of the shake flask broth was used to inoculate a 2 liter fermentationvessel.

A total of 1.8 liters of the fermentation batch medium was added to atwo liter glass jacketed fermentor (Applikon Biotechnology, Schiedam,Netherlands). Fermentation feed medium was dosed at a rate of 4 g/l/hourfor a period of 72 hours. Fermentation batch medium was composed of 5 gof yeast extract, 176 g of powdered cellulose, 2 g of glucose, 1 g ofNaCl, 1 g of Bacto Peptone, 4 g of K₂HPO₄, 0.2 g of CaCl₂.2H₂O, 0.2 g ofMgSO₄.7H₂O, 2.5 g of citric acid, 5 g of NH₄NO₃, 1.8 ml of anti-foam, 1ml of trace elements solution, and deionized water to 1 liter. Thefermentation vessel was maintained at a temperature of 45° C. and pH wascontrolled using an Applikon 1030 control system (ApplikonBiotechnology, Schiedam, Netherlands) to a set-point of 5.6+/−0.1. Airwas added to the vessel at a rate of 1 vvm and the broth was agitated byRushton impeller rotating at 1100 to 1300 rpm. At the end of thefermentation, whole broth was harvested from the vessel and centrifugedat 3000×g to remove the biomass.

Example 2 Purification of Myceliophthora thermophila CBS 117.65Cellobiose Dehydrogenase

The Myceliophthora thermophila CBS 117.65 harvested broth described inExample 1 was centrifuged in 500 ml bottles at 13,000×g for 20 minutesat 4° C. and then sterile filtered using a 0.22 μm polyethersulfonemembrane (Millipore, Bedford, Mass., USA). The filtered broth wasconcentrated and buffer exchanged with 20 mM Tris-HCl pH 8.5 using atangential flow concentrator (Pall Filtron, Northborough, Mass., USA)equipped with a 10 kDa polyethersulfone membrane (Pall Filtron,Northborough, Mass., USA). To decrease the amount of pigment, theconcentrate was applied to a 60 ml Q-SEPHAROSE BIG BEAD™ column (GEHealthcare, Piscataway, N.J., USA) equilibrated with 20 mM Tris-HCl pH8.5, and eluted stepwise with equilibration buffer containing 600 mMNaCl. Flow-through and eluate fractions were analyzed by SDS-PAGE using8-16% CRITERION™ SDS-PAGE gels (Bio-Rad Laboratories, Inc., Hercules,Calif., USA) and stained with GELCODE™ Blue protein stain (Thermo FisherScientific, Waltham, Mass., USA). The eluate fraction containedcellobiose dehydrogenase (CBDH) as judged by the presence of a bandcorresponding to the apparent molecular weight of approximately 100 kDaby SDS-PAGE (Schou et al., 1998, Biochem. J. 330: 565-571).

The eluate fraction was concentrated using an AMICON™ ultrafiltrationdevice (Millipore, Bedford, Mass., USA) equipped with a 10 kDapolyethersulfone membrane, and buffer-exchanged into 20 mM Tris-HCl pH8.5 using a HIPREP® 26/10 desalting column (GE Heathcare, Piscataway,N.J., USA). The desalted material was loaded onto a MONO Q® column (HR16/10, GE Healthcare, Piscataway, N.J., USA) equilibrated with 20 mMTris-HCl pH 8.5. Bound proteins were eluted with a linear NaCl gradientfrom 0 to 500 mM (18 column volumes) in 20 mM Tris-HCl pH 8.5. Fractionswere analyzed by SDS-PAGE as described above and the cellobiosedehydrogenase eluted at approximately 350-400 mM NaCl.

Fractions containing cellobiose dehydrogenase were pooled (60 ml) andmixed with an equal volume of 20 mM Tris-HCl pH 7.5 containing 3.4 Mammonium sulfate to yield a final concentration of 1.7 M ammoniumsulfate. The sample was filtered (0.2 μM syringe filter,polyethersulfone membrane, Whatman, Maidstone, United Kingdom) to removeparticulate matter prior to loading onto a Phenyl Superose column (HR16/10, GE Healthcare, Piscataway, N.J., USA) equilibrated with 1.7 Mammonium sulfate in 20 mM Tris-HCl pH 7.5. Bound proteins were elutedwith a decreasing 1.7→0 M ammonium sulfate gradient (12 column volumes)in 20 mM Tris-HCl pH 7.5. Fractions were analyzed by SDS-PAGE asdescribed above and the cellobiose dehydrogenase eluted at approximately800 mM ammonium sulfate. The cellobiose dehydrogenase fraction was >90%pure as judged by SDS-PAGE. CBDH activity was confirmed by a2,6-dichlorophenolindophenol (DCIP) reduction assay in the presence ofcellobiose, as described by Schou et al., 1998, supra.

Fractions containing cellobiose dehydrogenase were pooled, concentrated,and buffer exchanged into 20 mM Tris-HCl pH 7.5 by centrifugalconcentration in a SORVALL® RT7 centrifuge (Thermo Fisher Scientific,Waltham, Mass., USA) using VIVASPIN™ 20 centrifugal concentrators. (10kDa polyethersulfone membrane; Sartorius, Göttingen, Germany) at 1877×g.Protein concentration was determined using a Microplate BCA™ ProteinAssay Kit (Thermo Fischer Scientific, Waltham, Mass., USA) in whichbovine serum albumin was used as a protein standard.

Example 3 Pretreatment of Corn Stover

Corn stover was pretreated at the U.S. Department of Energy NationalRenewable Energy Laboratory (NREL) using dilute sulfuric acid. Thefollowing conditions were used for the pretreatment: 1.4 wt % sulfuricacid at 165° C. and 107 psi for 8 minutes. According to NREL, thewater-insoluble solids in the pretreated corn stover (PCS) contained56.5% cellulose, 4.6% hemicellulose and 28.4% lignin. Cellulose andhemicellulose were determined by a two-stage sulfuric acid hydrolysiswith subsequent analysis of sugars by high performance liquidchromatography using NREL Standard Analytical Procedure #002. Lignin wasdetermined gravimetrically after hydrolyzing the cellulose andhemicellulose fractions with sulfuric acid using NREL StandardAnalytical Procedure #003. The PCS was washed with a large volume of DDIwater on a glass filter.

Example 4 The Effect of Anaerobicity on CellobioseDehydrogenase-Inhibited PCS Hydrolysis

The effect of anaerobicity on hydrolysis of PCS was evaluated in thepresence of Humicola insolens cellobiose dehydrogenase (CBDH) orMyceliophthora thermophila CBS 117.65 cellobiose dehydrogenase.

The hydrolysis of PCS was conducted using 125 ml Erlenmeyer flaskscontaining a total reaction mass of 50 g. The hydrolysis was performedwith 16.5% total solids of unwashed, whole slurry PCS, equivalent to47.85 mg of cellulose per ml, in 50 mM sodium acetate pH 5.0 buffercontaining 1 mM manganese sulfate and a Trichoderma reesei cellulasecomposition (CELLUCLAST® supplemented with Aspergillus oryzaebeta-glucosidase available from Novozymes A/S, Bagsværd, Denmark; thecellulase composition is designated herein in the Examples as“Trichoderma reesei cellulase composition”) at 4 mg per g of PCS.Cellobiose dehydrogenase was added at concentrations between 0 and 10%(w/w) of total protein. Flasks were incubated at 50° C. for 72-168 hourswith shaking at 150 rpm. All experiments were performed in triplicate.

At various time points between 24 and 168 hours of incubation, 100 μlaliquots were removed and the extent of hydrolysis was assayed byhigh-performance liquid chromatography (HPLC) using the protocoldescribed below.

To establish anaerobicity, buffer and PCS were added in 125 mlErlenmeyer flasks and bubbled with gaseous nitrogen (N₂) forapproximately 5 minutes prior to enzyme addition in an environment bag(AtmosBag, Sigma Chemical Co., St. Louis, Mo., USA) under positive N₂pressure (ultrapure N₂, Airgas). The Erlenmeyer flasks were then sealedwith air-tight glass caps (Chemglass, Vineland, N.J., USA) using vacuumgrease and secured with a wire cage. The reaction flasks were allowed toequilibrate at 50° C., returned to the N₂ environment, vented to releasegas pressure, and resealed. Hydrolysis was allowed to proceed at 50° C.for 72-168 hours. At various time points during the hydrolysis, theflasks were returned to the nitrogen environment and equilibrated atroom temperature, and then 100 μl aliquots were removed and filtered.The soluble fractions were analyzed by HPLC. Suction was noted when capswere removed, indicating that an air-tight seal had been maintained overthe course of the hydrolysis. In later experiments, anaerobic sensorstrips (BD Bioscience, Sparks, Mo.) were affixed to the inside of thecaps and confirmed that anaerobicity was generally maintained.

For HPLC analysis, samples were filtered with a 0.45 μm MULTISCREEN®96-well filter plate (Millipore, Bedford, Mass., USA) and filtratesanalyzed for sugar content as described below. When not usedimmediately, filtered aliquots were frozen at −20° C. The sugarconcentrations of samples diluted in 0.005 M H₂SO₄ were measured using a4.6×250 mm AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules,Calif., USA) by elution with 0.5% w/w benzoic acid-5 mM H₂SO₄ at a flowrate of 0.6 ml per minute at 65° C. for 11 minutes, and quantitation byintegration of glucose and cellobiose signal from refractive indexdetection (CHEMSTATION®, AGILENT® 1100 HPLC, Agilent Technologies, SantaClara, Calif., USA) calibrated by pure sugar samples. The resultantequivalents were used to calculate the percentage of celluloseconversion for each reaction. The extent of each hydrolysis wasdetermined as the fraction of total cellulose converted tocellobiose+glucose, and were not corrected for soluble sugars present inPCS liquor.

All HPLC data processing was performed using Kaleidagraph software(Synergy software, Reading, Pa., USA). Measured sugar concentrationswere adjusted for the appropriate dilution factor. Glucose andcellobiose were chromatographically separated and integrated and theirrespective concentrations determined independently. However, tocalculate total conversion the glucose and cellobiose values werecombined. Fractional hydrolysis is reported as the overall massconversion of [glucose+cellobiose]/[total cellulose]. Triplicate datapoints were averaged and standard deviation was calculated.

The results (FIG. 1A) demonstrated that the Trichoderma reesei cellulasecomposition hydrolyzed PCS equally well whether under anaerobic oraerobic conditions. However, PCS hydrolysis in the presence of 5%Thermoascus aurantiacus GH61A polypeptide under anaerobic conditions wasgreater than that observed under aerobic conditions. The overallhydrolysis was improved approximately 3%.

The addition of Myceliopthora thermophila cellobiose dehydrogenaseresulted in a 2% inhibition of PCS hydrolysis under aerobic conditions,and no inhibition under anaerobic conditions. Furthermore, thehydrolytic enhancement arising from GH61 addition was greater underanaerobic conditions than observed for aerobic hydrolysis.

FIG. 1B shows the kinetics of hydrolysis by 8 mg of the Trichodermareesei cellulase composition per g cellulose plus 8 mg of cellobiosedehydrogenase per g cellulose demonstrating the higher extent ofhydrolysis and the higher extent of CBDH inhibition. The dataillustrated the reduction in cellobiose dehydrogenase inhibition byperforming the hydrolysis under anaerobic conditions.

The addition of 0.4 mg of Humicola insolens cellobiose dehydrogenase perg cellulose to a hydrolysis reaction of 50 g of whole slurry pretreatedcorn stover by 4 mg of the Trichoderma reesei cellulase composition perg cellulose under anaerobic conditions was approximately 10% lessinhibitory than a similar reaction performed under aerobic conditions(FIG. 1B).

Example 5 Effects of Anaerobicity on Hydrolysis of Pretreated CornStover (PCS)

Hydrolysis of PCS under anaerobic conditions was performed as describedin Example 4 with the following changes. Saccharification reactions werebubbled with N₂ for 5-minutes, adding the Trichoderma reesei cellulasecomposition and sealing the samples using vacuum-greased glass caps(Chemglass, Vineland, N.J., USA) and affixing wire caging to preventloss of seal arising from gas expansion at higher temps, in aN₂-inflated environment bag (AtmosBag, Sigma Chemical Co., St. Louis,Mo., USA) prior to incubation at 50° C. Fifty gram reactions wereperformed as described in Example 4 using various Trichoderma reeseicellulase composition concentrations as indicated. Reaction progress wasassayed by removal of 200 μl aliquots for HPLC analysis from theErlenmeyer flasks daily under N₂ within the environment bag, andresealing. HPLC analysis was performed as described in Example 4.

The results (FIG. 2) demonstrated that PCS hydrolysis under anaerobicconditions yielded higher fractional hydrolysis at all enzymeconcentrations and at all time points. The effect was most pronouncedbetween 4 and 7 days of hydrolysis, and at the intermediate enzymeconcentration of 8 mg per g cellulose. After 7 days of hydrolysis, theanaerobic enhancement over aerobic hydrolysis was 14-26%. The amount ofenzyme required for 80% conversion of cellulose to glucose andcellobiose in 7 days was reduced from approximately 9 mg per g celluloseunder aerobic conditions to approximately 5 mg per g cellulose underanaerobic conditions.

Example 6 Effect of Anaerobicity on Iron-Dependent Cellulase Inhibition

A series of 50 g scale hydrolyses of 25 g of AVICEL® (Sigma Chemical Co,St. Louis, Mo., USA) per liter by 0.25 g of the Trichoderma reeseicellulase composition per liter, with or without 0.025 g of Thermoascusaurantiacus GH61A polypeptide per liter and 1 mM FeSO₄, in 50 mM sodiumacetate pH 5.0 at 50° C., was performed under aerobic and anaerobicconditions as described in Example 5.

The results (FIG. 3) demonstrated that FeSO₄ caused approximately a 30%decrease in the normal aerobic hydrolysis after one day, but not in theanaerobic hydrolysis. In the absence of FeSO₄, slightly higherhydrolysis was also observed under anaerobic conditions.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention.

Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description. Such modifications are also intendedto fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

1. A method for pretreating a cellulosic material, comprising:pretreating the cellulosic material under anaerobic conditions toproduce a pretreated cellulosic material, wherein the pretreating of thecellulosic material is a chemical pretreatment and the anaerobicconditions are an oxygen level of no greater than 0.025 mMwater-dissolved oxygen.
 2. The method of claim 1, further comprisingrecovering the pretreated cellulosic material.
 3. The method of claim 1,wherein the chemical pretreatment is selected from the group consistingof steam pretreatment with explosion, steam pretreatment withoutexplosion, dilute acid pretreatment, hot liquid water pretreatment,alkaline pretreatment, lime pretreatment, wet explosion, ammonia fiberexplosion, organosolv pretreatment, ammonia percolation pretreatment,ultrasound pretreatment, electroporation pretreatment, microwavepretreatment, supercritical CO₂ pretreatment, supercritical H₂Opretreatment, and gamma irradiation pretreatment.
 4. The method of claim1, wherein the anaerobic conditions are an oxygen level of no greaterthan 0.01 mM water-dissolved oxygen.
 5. The method of claim 1, whereinthe anaerobic conditions are an oxygen level of no greater than 0.002 mMwater-dissolved oxygen.
 6. The method of claim 1, wherein the anaerobicconditions during pretreatment increases the hydrolysis of thecellulosic material during saccharification compared to aerobicconditions during pretreatment.
 7. The method of claim 1, wherein theanaerobic conditions are an oxygen level of no greater than 0.001 mMwater-dissolved oxygen.
 8. A method of obtaining a fermentable productfurther comprising: (a) saccharifying the pretreated cellulosic materialobtained by the method of claim 1 with an enzyme composition; (b)fermenting the saccharified cellulosic material with one or morefermenting microorganisms to produce a fermentation product; and (c)recovering the fermentation product from the fermentation.
 9. The methodof claim 8, wherein the enzyme composition comprises one or morecellulolytic enzymes, one or more xylan degrading enzymes, or one ormore cellulolytic enzymes and one or more xylan degrading enzymes. 10.The method of claim 9, wherein the one or more cellulolytic enzymes areselected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.
 11. The method of claim 10,wherein the enzyme composition further comprises a polypeptide havingcellulolytic enhancing activity.
 12. The method of claim 9, wherein theone or more xylan degrading enzymes are selected from the groupconsisting of a xylanase, an acetyxylan esterase, a feruloyl esterase,an arabinofuranosidase, a xylosidase, a glucuronidase.
 13. The method ofclaim 8, wherein step (a) and step (b) are performed simultaneously. 14.The method of claim 8, wherein the saccharification is performed underanaerobic conditions at an oxygen level of no greater than 0.025 mMwater-dissolved oxygen.
 15. The method of claim 14, wherein thesaccharification is performed under anaerobic conditions at an oxygenlevel of no greater than 0.01 mM water-dissolved oxygen.
 16. The methodof claim 15, wherein the saccharification is performed under anaerobicconditions at an oxygen level of no greater than 0.002 mMwater-dissolved oxygen.
 17. The method of claim 8, wherein thefermentation product is an alcohol, an amino acid, a ketone, a gas, oran organic acid.
 18. The method of claim 16, wherein thesaccharification is performed under anaerobic conditions at an oxygenlevel of no greater than 0.001 mM water-dissolved oxygen.